Science method

Cytology - Science method

Explore the latest questions and answers in Cytology, and find Cytology experts.
Questions related to Cytology
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I am doing an analysis of a dataset. And I want to find out if cytological features can predict/correlate well with acid fast bacilli positivity/negativity. I know such work has been but I am considering using multiple logistic regression analysis.
Predictor = cytology
Outcome = Positive AFB
I am not sure if multiple logistic regression analysis tool is the best statistical tool to use for this prediction model. I would appreciate any feedback or comments.
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I am new to R so I have a steep learning curve.
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These are the microscopic images of a fruit pulp observed under Binocular microscope at 40x.
Please help me identify the same.
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I believed that the spring shape is the xylem of the plant
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Hello,
We are planning to collect and centrifuge mice bronchoalveolar lavage fluid cells. The downstream analysis we are planning to do (neutrophils, macrophage and lymphocyte % distribution analysis) requires us to transport the cells to another laboratory while maintaining viability. How are the cells normally transported? In what medium, what temperature? How long could the cells be stored?
Thank you!
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In my opinion, you should not add anything, take the lavage and place in appropriate size tube with minimum PBS just to help transfer lavage to the tube. Transfer the tube at room temperature. Start processing right away at arrival.
I have done with bone arrow (heparinized), upto 4 hours at room temperature, all cells were fine.
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I would like to ask whether there will be a worse result of the immunocytochemical reaction, which will be carried out on archival materials (2006-2008): cytological smears of lung cancer, stained by Pappenheim and Papanicolaou?
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I am afraid it won't work, especially on the previously stained smears.
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Dear researchers, according to your practice in field of staining bone marrow cells in rodents.
What are the types of staining and which is the best and easy used differentiating BM cytology.
Dose anyone have atlas or reference guide for rats bone marrow cytology?
Thanks in advance
Dr. Ali Alchalabi
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If we want to detect infectious agents, there are various histological stains for the agent. however, if we want to look for different cells, we can use immunohistochemical stains. Which different cells do you want to detect?
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Do you think it is possible to study the spread of lung cancer in the parenchyma only by cytological and immunocytochemical methods (if access to histological material is limited)?
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Kindly check also the following good RG link:
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Hello! Help me, please. I have a problem with washing away cells from cytological smears...
Currently, our laboratory uses a manual method of staining cytological smears with immunocytochemical dyes:
1) Fix cytological smears in methyl alcohol for 5 seconds.
2) Stain cytological smears by Pappenheim and Papanicolaou methods.
3) Dissolve 0.01 grams of trypsin in 1 ml of phosphate-buffered saline (PBS) (PH 7.2-7.4) and apply to smears for 10 seconds. (!!!!!) (Are there any errors at this stage? Probably we need to dissolve 0.001 grams of trypsin in 100 microliters PBS? Or reduce the exposure time of the solution from 10 seconds?)
4) Thoroughly wash the smears with PBS.
5) Wash the smears with distilled water to prevent drying.
5) Apply to smears 5% acetic acid solution for 10-15 minutes.
7) Wash the smears again with distilled water.
8) Apply to smears 1% hydrogen peroxide solution for 30 minutes.
9) Thoroughly wash the smears with PBS.
10) Apply the first (І) antibodies to the smears for 1.5 hours (at room temperature or at -4 C per day) in a humid chamber.
11) Wash thoroughly with PBS.
12) Apply the second (ІІ) antibody to the smears: Mix 1 microliter of the second antibody, 10 microliters of human serum 4 (AB) group RH (-) and 100 microliters of PBS and apply to smears for 1.5 hours at room temperature.
13) Thoroughly wash the smears with PBS.
14) Mix 1 milligram of 3 diaminobenzidine tetrachloride (DAB), 6 milliliters of PBS, and 20 microliters of 6% hydrogen peroxide solution and apply to cytological smears for 15 minutes. 15) Thoroughly wash the smears with distilled water.
Please indicate what I am doing wrong, or advise where you can find a better manual technique in which the cells will not be washed away from the cytological smears.
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Use superfrost plus slides for your smears and take care that they are not too thick. I think the protein digestion is the most critical point in your protocol and the reason why the smears are washed away during the immuno process. If the trypsin pretreatment is necessary I would recommand to prepare a stock solution. Play with different concentrations of the trypsin solution and incubation times. Sometimes a permiabilisation with Triton-x-PBS (0,2% Triton in PBS for 30 min; 3 x 5 min rinsing in PBS afterwards) could be sufficient.
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Tell me please, which of p63 antibodies (Manufacturer "Diagnostic biosystems": rabbit RMAB086-01 (antibody titer 1:50); mouse BSB 3605 (antibody titer 1:200) ; mouse BSB3602) is better for staining cytological smears of alveolocyte II type and cells of lung cancer (adenocarcinoma, squamous cell cancer)?
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Rabbit provides greater benefit over mice when it comes to monoclonal antibodies. Immunoglobulin genes from rabbits form antibodies that fit a much wider range of epitopes than mouse antibodies. Isoforms of a protein that differ by one amino acid residue, results in slight variations in structure. The rabbit's immune system better recognizes these subtle differences, producing monoclonal antibodies.
Moreover, in immunodominance certain epitopes from the same antigen are more immunogenic than others thereby the immune system produces a great amount of antibodies against the dominantly immunogenic epitope, and few antibodies against the other epitopes. In this case, rabbits exhibit less immunodominance than mice.
So, rabbit monoclonal antibody because of the unique features of the rabbit immune system would be more preferred than the mouse monoclonal antibody.
You could also refer to the research article below wherein rabbit monoclonal was preferred over mouse monoclonal antibody for ICC.
So, I recommend that you use p63 rabbit monoclonal antibody for immunocytochemistry in your study because of its higher affinity and increased specificity.
Good Luck.
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Hello everyone, I want to know if there is a golden technique in the diagnosis of canine leishmaniasis to evaluate the results of other diagnostic techniques such as ELISA and IFAT and even lymph node or splenic cytology in the latter.
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urine sedimentation technique
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"Approximately 15–30% of all thyroid nodules evaluated with fine-needle aspiration biopsy (FNAB) are classified as cytologically indeterminate. The stepwise unraveling of the molecular etiology of thyroid nodules has provided the basis for a better understanding of indeterminate samples and an opportunity to decrease diagnostic surgery in this group of patients."
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Recently, TBSRTC, 2nd ed. has been using and Category III, atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS); IV, follicular neoplasm (FN) or suspicious for a follicular neoplasm (SFN); and V, suspicious for malignancy (SM) have been accepted as constituting indeterminate cytology of thyroid nodules. The risk of malignancies (ROMs) for indeterminate cytology are declared as 5-15%, 15-30%, and 60-75% in TBSRTC 1st ed. while 10-30%, 25-40%, and 50-75% in 2nd ed. for Category III, IV, and V, respectively, reflecting a wide range of ROM. The 2016 Royal College of Pathologists, United Kingdom (RCPath, UK) reported indeterminate cytology as Thy 3a, neoplasm possible, atypia/non-diagnostic and Thy 3f, neoplasm possible, suggesting FN, with the ROMs of 20-31% and 24-39%, respectively. In addition, the 2016 RCPath, UK declared Thy 4, SM with the ROM of 70-87%. The 2014 Italian Consensus for the Classification and Reporting of Thyroid Cytology (ICCRTC) divided diagnostic category TIR3, indeterminate cytology, into two subcategories, TIR3A (low-risk indeterminate lesion) and TIR3B (high-risk indeterminate lesion), with different expected ROMs and discrete clinical manners as <10% and 15-30%, respectively. The 2014 ICCRTC also harbors TIR4, SM with an expected ROM of 60-80%. The 2014 Royal College of Pathologists of Australasia (RCPA) and the Australian Society of Cytology (ASC) propounded: i) 3, Indeterminate (AUS/FLUS) with low ROM, 5-13%; ii) 4, Suggestive of an FN (FN/SFN) with moderate ROM, 21-26%; and iii) 5, SM with high ROM, 85-90%. The 2013 Japan Thyroid Association (JTA) Guideline for the Management of Thyroid Nodules declared: i) 3, Indeterminate, 3B, others; Indeterminate, ii) 3A, FNs, 3A-1, favor benign, 3A-2, borderline, 3A-3, favor malignant; iii) 4, Malignancy suspected, respectively. As such, each diagnostic category of the last TBSRTC, 2nd ed., with noninvasive follicular thyroid neoplasm with papillary-like nuclear features, NIFTP, harbor a wide range of implied ROMs.
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What is your distinctive clinical approaches for the management of thyroid nodules of both 10-15 mm and over 15 mm, separately, with Category III of indeterminate cytology, TBSRTC, 1st and 2nd ed., harboring high-to-intermediate suspicion sonographic pattern, low clinical risk factors, repeated FNA cytology, molecular testing, or both, are not performed or inconclusive?
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We might recommend surveillance for the management of the mentioned thyroid nodules, 10-15 mm with Category III of indeterminate cytology, TBSRTC, 1st and 2nd ed., harboring high-to-intermediate suspicion sonographic pattern, low clinical risk factors, repeated FNA cytology, molecular testing, or both, are not performed or inconclusive, comparing the ones >15 mm. This issue deserves further investigation.
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I am so curious how ideogram was constructed. I found a publication that used software to construct ideogram, but this software seems like a photoshop software. Is there any proper software to draw or construct an ideogram? thanks ^^
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For drawing Ideogram you can Visio software as well. It is very easy and applicable. Below is the link if you want to download and use it, but you have to purchase and activate it for the full version. Fortunately, I have the crack and if you are interested I can give it to you.
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We are examining tissue from a cluster of human and canine patients with a similar pattern of systemic illness of unknown cause. All members of the cluster have evidence of motile zoospore-like objects in their blood and other tissue aspirates. Control wet-mount preps from healthy relatives of these patients do not show the presence of such motile objects.
Despite the tiny size of these motile objects, the “swimming” motion seems more consistent with the “falling leaf” forward motility pattern associated with a eukaryotic flagella than with bacterial motility patterns. Also, the staining patterns and SEM appearance of these objects appears more consistent with a eukaryote.
Preliminary sequencing studies have suggested sequence homology with stramnopile-type organisms. We are attempting to sequence cultured colonies of the organism but are having extremely low DNA yields despite robust growth of the organism in culture.
We would appreciate the opinion of those familiar with the morphology and zoospore motility of oomycete and related type organisms about the similarities and differences seen in these movies of motility in unstained, aseptically collected adipose tissue nodules from a patient in this cluster, suspected to be infected with a novel or emerging type of eukaryotic pathogen.
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sure, I'd like to see the pictures - philageis@aol.com
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I'm used standard squash technique using fuelgen staining.
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Nice! Good question....yea..if we know techniques of your experimental, I may tell my students to repeat the same,,, regards!
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Hello, Our background is explained in the profile section- but we are a group of veterinarians/other scientists working on an unexpected research project that arose from observations of a cluster of illnesses among dogs and their owners in our veterinary practice. After thorough traditional work-up, we ruled out known causes of this constellation of symptoms and because of the seemingly transmissible nature of the illness, we began looking for perhaps a rare infection. We did find unexplainable objects in urine, cyst fluid, and subcutaneous nodules from the affected animals/humans- and did not see those same objects in blinded control studies using spouses/housemates of the affected individuals. It has taken a while to characterize the shape/properties of these objects since we were seeing them in the context of semi-degraded in tissue or tangled/broken up in urine samples. But now, we have retrieved enough reasonably intact samples to realize that they are large shell-shaped objects, resembling bivalves slightly, that contain long thin complex fibers as structural elements. The hinge/latch-like objects along the length of the fiber appear to bind to other elements in the internal contents of the "shell" and help keep them packaged and organized. I'm quoting "shell" because despite the marked resemblance to some bivalve shells, the shell portion is more dynamic. It can stretch and when the shell opens and the internal contents unfurl, the majority of the tissue forming the "shell" shape actually comes away in plumes of sheets of tissue that unfold in a very organized manner. Intriguingly, these sheets of tissue appear to contain a middle layer with a non-staining fibrous semi-liquid filling that may be mesoglea. The sheets of tissue are made up of connected small versions of almost exactly the same shell body plan as the larger organisms, and these small objects appear connected by a series of tubes that resemble stolons- leading us to wonder if the organism could be some type of hydrozoa or even myxozoan? I've attached a few new photos to demonstrate. We would appreciate any opinions at all about what the identity of these objects may be, and how we can optimize staining protocols +/- DNA extraction techniques to be able to get an identifying sequence for these probable organisms- though there will undoubtedly be some contamination with human/canine DNA.
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Yes..it seems some invertebrates
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Hello, there is more background in our overview page, but briefly- we are a group of veterinarians who came across unusual microscopic findings in a group of dogs and their owners (who happened to also be veterinarians) with an otherwise unexplained syndrome involving hematuria, chronic fibrosis of deep connective tissues, chronic cough, fatigue, and neuropathy. Both the dogs and their owners had a thorough medical work-up in which the cause of illness cold not be determined. Several other close human contacts of the original humans/dogs affected also developed similar symptoms over the following year. Some of the humans have had plateau of their symptoms while others have continued to worsen. The structures shown in attached images were found in the urine, blood, needle aspirates of subcutaneous nodules, and sometimes sputum of the affected individuals (canine and human) and not in healthy family members (matched controls)- this finding was confirmed by blinded readings of the affected vs. control cytology samples. CDC has been informed of these findings and may begin an investigation, though they are hobbled by the current COVID crisis and also relayed that they do not have a pathologist on staff trained to read this type of cytology!? So, we are asking your help in determining if the objects shown do seem to match the criteria for some type of protist (or other organism?), or if they are some type of unusual artifact. We have attempted sequencing without success, but this may be hampered by a thick glue-like mucus that seems to be produced by the organisms binding everything in the near vicinity tightly together. As veterinarians, our knowledge of invertebrate zoology is limited- but collectively, we thought that the objects seemed to resemble cysts of amoebae or perhaps ciliates, or even myxozoans. They are protected by a very stain-resistant outer "shell" (test?) that seems made of aggregates of regional debris. However, some layers stain well with Alcian Blue/Aniline Blue and negative staining with Nigrosan helped to clarify the appearance of some of the objects. The white surface is very birefringent, so we were only able to get clear micrographs when stacking software was used to improve focal range. Some features seemed similar to entamoeba histolytic, but the outer cyst wall is too thick in many examples. Some resembled Blastocystis- with a large central vacuole, but again, there were inconsistencies with that identification as well. If anyone can point out specific details/features that suggest real organism vs artifact, and/or label any of the details for us to give us some landmarks to follow (we though we were seeing distinct nuclei, but were not certain...) that would be extremely helpful.
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Hi
Photos show artifacts and no structures seen at all related to a protozoan, all protozoa usually have regular structures in various forms. Search the slide again carefully and also with high magnification!
good luck
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Hello everyone,
I am looking for a scientist who have experance in histopathology and cytology to advise me the essential equipments that using in Histopathology and cytology Laboratory. We want to establish this Laboratory in our new centre for cancer treatment.
THANK YOU
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Good day,
Equipment of Histopathology and Cytology lab depends from volume of biopsies which is planning to be, the clinical departments etc. I can give you some advises about Histopathology equipment, in my country Histopathology and Cytology are different departments.
Best regards,
Dmitry
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In a patient with multinodular goitre ( non toxic and no compressive symptoms) , with a single suspicious nodule (2 cm) on ultrasonography and cytology of that nodule as Bethesda V ( suspicious of papillary carcinoma), what would the extent of initial surgery be? Hemithyroidectomy? Total thyroidectomy?
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If the contralateral lobe is normal and is not an unfavorable histological variety, for a lesion of that size both procedures are acceptable
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Full history is in the project description, but briefly- a team of other veterinarians/colleagues and myself have stumbled across a case of multiple animals that have similar symptoms and are co-housed. Their presentation suggests an infectious disease, but no known infectious agent could be found despite thorough work-up. However, cytology samples from clean aspirates of subcutaneous nodules and urine filtrate (they have bloody urine) seems to be showing repeated structures that do not appear mammalian in origin. We seek to understand if these structures are some type of very organized artifact, or if they instead suggest that there may be some very unusual/novel type of organism (Annelid or Polychaete esp.) causing/involved-in these animals illnesses. Thank you for your time!
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@ Matthew B Paddock; Thanks for your reply. We did attempt 16s sequencing of a tissue sample taken from one of the subcutaneous nodules (before we realized that there could be a eukaryote involved). The 16S sequencing (from RNA extracted from tissue blocks from what should have been a surgically sterile site) did show a mixture of many bacterial species. However, the profile wasn't anything that would suggest a relative of a known pathogen, nor did it suggest surface contamination (ie- no staph species etc..) The profile didn't have any one main dominant species either, but was 20% this, 18% that, 15% this etc.... I don't have the profile in front of me right now, but, from memory, the bacterial species in the tissue sample were unusual, with an overrepresentation of extremophiles. At first I interpreted this as potentially some species that might be surviving our autoclaving of instruments (ie- could tolerate the autoclave temperatures but not typically mammalian pathogens so were not the cause of disease in the animals). But, once the observation of the filaments/worm-like organisms was made, it did occur to me to that perhaps if the infection were eukaryotic, perhaps the profile of bacteria seen could reflect the composition of the microbiome of the putative novel parasite. I cross-referenced the profile of bacteria with the known microbiome profile of c-elegans and, though not an exact match, the proportions and types of bacterial species represented did suggest that perhaps the species identified could represent the bacterial population of some eukaryotic species. Our first thought was that the putative novel species was some type of nematode/filarial infection. We did attempt to have the tissue sample probed with nematode-specific 18S rRNA primers to see if we could amplify/sequence any nematode DNA. Interestingly, the primers did bind and amplify a target sequence, but when that was attempted to be sequenced, the PCR products were not clean enough to yield an identifiable species. Later, other microscopic findings led us to consider other phyla (beyond nematodes/trematodes). Since this is mostly a project done out of curiosity rather than our primary line of work, we have not had the budget to proceed with any additional PCR studies. If you would like to see the actual genus/species break-down for the 16S analysis, I'd be happy to dig it up and post it for you (just don't have access to that computer today).
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Hi. As far as I understand you just be having histology as your gold standard. What I want to understand is what were your true negatives ? Say if p16 and ki 67 came out negative on cytology how did you confirm it's a true negative? Did you have another negative group?or you took the same histology as negative control too?
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I am reading your comment here now.
Yes, Nikhat. Both Tauangtham and I have understood your question the first time around.
It seems though that you did not read carefully the scientific links that I have sent you.
If you had, you would have understood that it is long established that there is no such thing as a 100% fool-proof diagnosis of benign, pre-cancerous or cancerous histology, based on three imperfect tests, the scope of which (esp. sensitivity & specificity) we still explore, and that are, therefore combined to complement each other.
In this format (and without knowing the life style, the medical history or the current physiology of the women that are being tested), these three tests used once will never give a 100% negative testing results. Your supervisor is correct.
You are testing living creatures with complex anatomy and physiology, whose body condition changes dynamically. Anomalous cytology is present at all times, but some of it is successfully cleared by the immune system, while other is not.
It takes time for persistent anomaly to develop to a degree sufficient to diagnose in histology.
Testing negative for diffuse p16 today does not automatically mean health, or that the same woman is going to test negative for diffuse p16 again in three-months' time:
Diagnosis would also depend on lab guidelines, as well as patho-histologist's personal judgement and experience.
The last link I have sent you is a dissertation that discusses new DNA methylation markers to try and better the success rate of the three testing tools you work with.
As a physician, I will argue that (and I have already told you via messaging) in the face of:
-- a pronounced lack of certainty on how many diseases develop, and
- imperfect test data on patients' condition,
it is unethical (to recommend to patients or) not to argue in favor of exploring all avenues (even invasive ones) to:
- make an informed diagnosis, (and by doing so)
- save patients more anxiety, suffering and stronger medication (with negative side-effects) due to delayed/incorrectly made diagnosis, and
- ensure that patients stay alive (by detecting pre-cancerous and cancerous conditions early)
I hope this brings further clarification.
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Morphological, cytological, biochemical and molecular tools are used to differentiate Two species. Which is the best and accurate method?
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I agree with Valeria Tananska , the reasoning being that morphological differentiation has been utilized exclusively, until recently. However, these days, molecular methods can provide exact quantification of the relatedness between two organisms. The issue here is that the molecular methods are not as well developed and as common to practice as morphology. As molecular methods become more commonplace within organism differentiation we will see a different, and hopefully more exact, classification of species. This does also mean that different classification methods might showcase alternative features, and perhaps even the opposite of what you would expect, so the methods might diverge in interpretation.
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Is it possible to extract DNA for further analysis/diagnosis from Papanicolaou stained cytology slide?
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Zohar gavish the Answer of Helena is correct
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Noninvasive follicular thyroid neoplasm with papillary like nuclear features is a very good topic for discussion.
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You can work with cytological specimens both retro- and prospective but (see the answer of Olena Polyakova) will need histological (surgical) material to make the diagnosis of NIFTP.
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Hello!
After daily vaginal lavages, we have had a mouse who appears to have been in estrus for three days in a row based off of cytology and the presence of only cornfield epithelial cells. The samples typically dry clear, but as you can see in the photo this one dried very white. Also, yesterday both of her samples washed off the slide after ample drying time and our standard 1% toluidine blue staining protocol.
Any idea what might be happening to this mouse in terms of her cycle?
Thank you in advance!
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it must be diluted or spread in order to be visualized well under microscope because it looks thick
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Many a times we don't get good cellularity to give a clear diagnosis of ovary in cytology. Any advise please?
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I still feel as and when Ovarian lesions are excised, one can prepare a set of good well fixed slides for study and learning from classical lesions.
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In literature there are different methods to detect hpv on bladder tumour studies. Frozen sections, direct tumour tissue, urine, cytology. Using PCR assay I'm planning a study on bladder tumour & hpv. Which sampling method do you recommend?
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actually I wanted to study on fresh samples. So urethral swab and first void samples I collected.
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Dear Sir/ Ma'am
Can any researcher share/provide the reprint of following articles (Avdulov 1928, 1931) on grass cyto-systematics...
1. Avdulov, N. P. (1928). Systematicheskaya kariologiya semeestva Gramineae. Drievnik vsesojuznogo Sezda Bot. Leningrade 1928: 65-66.
2. Avdulov, N. P. (1931). Karyo-systematische Untersuchungen der Familie Gramineen. Bull. Appl. Bot., Genet. & Plant Breeding Suppl. 43: 1-438.
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I can only provide you with Avdulov (1931) in Russian version (>270 pp), German version is not available.
The file is attached. You will need DJVU player software in order to open the file (free download available from https://www.cuminas.jp/en/downloads/download/?pid=1 )
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The taxonomy of the genus Crocus is extremely complicated due to the lack of clear distinctive characters, the wide range of habitats and the heterogeneity of the morphological traits and cytological data.
Moreover they show that there are several problems at the infraspecific level. Genetic dissimilarities between the known subspecies revealed that most of the subspecies must be ranked as species.
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The genus Crocus L. belongs to the large family Iridaceae and is a systematically problematic genus. In the Old World, about 100 species are known (Harpke et al., 2013). Although molecular studies have been increasingly used to examine the phylogeny of living organisms, they have only recently been applied to the genus Crocus (Petersen et al., 2008). DNA markers have been used to characterize germplasm collections, inform breeding programs, and facilitate genetic diversity studies and taxonomic analysis. The utilized methods include interretrotransposon amplified polymorphism (IRAP) (AlaviKia et al., 2008), random amplified polymorphic DNA (RAPD) (Grilli Caiola et al., 2004; Beiki et al., 2010; Rubio-Moraga et al., 2010), amplified fragment length polymorphism (AFLP) (Zubor et al., 2004; Erol et al., 2011; Nazzal et al., 2011), intersimple sequence repeat (ISSR) (Rubio-Moraga et al., 2010), and simple sequence repeat (SSR) (Rubio-Moraga et al., 2010; Nemati et al., 2012) analyses. Among these molecular markers, AFLPs were found to show high levels of polymorphism per primer pair and yield high resolution and reproducibility (Meudt and Clarke, 2007). Fore more information follow this link:
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anyone can advise the litrerature about methods(cytological,molecular and other ) of reserch orchid mycorriza?
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Dear collegues Elena & Georgy,
We have in Museum the group of Marc-André Selosse for orchid mycorrhiza,
in two aspects : évolution and ecology.
Он мастер по чтению лекций, но мне не все нравится в его работах по симбиозу.
Успехов
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Currently, I would like to study more about seed development and apomixis. I want to check the ploidy of endosperm of seed in the case apomixis happens. I know one way is to determine by using flow cytometry. But my lab is not keen on cytology. I wonder if there is another simple way or not? Thank you so much for reading my question.
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Dear Dung
In the attached papers, apomixis frequency and ploidy levels in Poa and Centotheca using flow cytometric techniques were examined, which I hope will be of interest to you.
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In this regards, I wish to point out that there is gross miss concept regarding soul & rebirth among almost all ethnic groups of human societies. I wish that people should rethink the concept of Rebirth & Germ Cell Soul & (not sole) & based on Science of Genetics-classical, cytological & molecular) the point of thinking is just similar as matter is not destroyed but transform into solid gas & liquid in the way germ cell egg & sperm) are not destroyed but continue the life through individuals female & male generation after generation. this phenomenon I call it "THE BIRTH''
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Kindly elaborate about the essence of your project. Are your findings against the soul theory mentioned in Geeta or Kriya Yoga techniqs....
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Protandim and NRF2 stimulator do that. Any researcher want to add some thing, I cordially invite them. I am very thankful to your suggestion.
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Does anyone have a recipe for Saccomanno fixative (a cytology fixative) for sputum preservation?
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Hello Hamdia,
Saccomanno's Fixative is an excellent pre-fixative for cytology specimens and the fixative of choice for collecting and fixing sputum specimens for cytology. This fixative in in fact, one of the most widely used fixatives in cytology that is recommended for cytology specimens such as sputums, urine, FNA'S, bronchial washings, pleural and peritoneal fluids, and ThinPrep™ preparations. The formula is accessable at the website indicated below:
Saccomanno's fixative is 50% ethyl alcohol which contains approximately 2% of Carbowax 1540 (Union Carbide Corporation, UCAR). Carbowax 1540 is solid at room temperature, with a melting point of 43 to 46 C. To avoid having to melt it whenever the fixative is prepared, a stock solution can be prepared by melting Carbowax (melted in an incubator or hot air oven at 50 to 100 C) and adding it to an equal volume of water or 50% ethyl alcohol. The mixture will not solidify. Saccomanno's fixative can then be prepared with 430 mL of water, 530 mL of 95% ethanol, and 40 mL of the stock Carbowax solution. Some light green SF or fast green FCF can be added to color the fixative. Koss warns that the denaturants in reagent alcohol may cause excessive hardening of mucus.
ref: Histology FAQ
Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan Department of Anatomy and Cell Biology The University of Western Ontario London, Canada
With best regards,
ROWEN T. YOLO, M.D.
Dept of Pathology
Faculty of Medicine and Surgery
University of Santo Tomas
Manila,Philippines
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My group is working on embryo rescue in Cucumis genus. We cultured the immature seeds and we got a lot of types of germination. Some seeds germinated as usual. But almost seeds regenerated callus. And I think we got two kinds of calli (embryogenic callus and non-embryogenic callus). The problem is that my lab is not expert in cytology or tissue culture. So that, I want to identify them in the simple way but correction. I am looking forward to your suggestions. Thank you in advances!
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Under the microscope the sotic emryo cells are round in shape. Highly cytoplasmic densely stained more over the starch grains are accumulated in the cytoplasma. See the attached file please
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the patient is 18 years old complains of weight loss, abdominal distention and menstrual irregularities. Is there a role for FNAC?
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Hello Dr Noha Hassan
I fully agree with the opinion of the others wherein correlation, with clinical presentation, biochemistry (AFP-B-HCG), and ultrasonographic findings point out that this is most likely a germ cell tumour of the ovary. In an 18 year old woman, this is most likely a mature cystic teratoma involving both ovaries which is not uncommon. The findings of ascitis and omentoperitoneal thickening may perhaps suggest peritoneal irritation with, in my opinion, mesothelial reactive changes that is secondary to the compression effects of the huge ovarian masses, however of course, I cannot discount the possibility of a malignant teratoma with peritoneal metastasis, albeit this is rare in this age group. Exploratory laparotomy with ovarian resection (or frozen section), peritoneal implant biopsy, and ascitic fluid cytology, would be the management of choice. In my opinion a pre-operative ultrasound-guided percutaneous FNAB may yield not much of a helpful information as far as diagnostic determination between a benign vs malignant teratoma is concerned. A sample of the ascitic fluid of course may be collected for cytodiagnosis, but may well prove to be of limited value in this case. I would be most interested to follow up this case. Please update me with any latest findings. Thank you very much for sharing this interesting case.
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Looking for ways to improve cell block for cytology.
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Hello, in my lab we use HistoGel - Thermo Scientific (Ref. HG-4000-012). The first spin (sample +fixative are made at 2800rpm (aprox. 1300-1500g) for 5 min. The second spin (sample + histogel)  3500rpm (2000-2250g) for 1 min.
Hope it helps
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I don't know how to adjust bonferroni correction to my data. 
Firstly, I tested the levels of several types of biomarker candidates in normal and cancer serum using ELISA. I found three types of potential biomarkers (A, B, C) for discriminating cervical cancer from normal cytology. 
Secondly, I combined three types of biomarkers using logistic regression model. 
Then, I wanna using bonferroni correction to reduce type I error but I have no idea how to adjust the bonferroni correction to my data. 
Is the bonferroni corrected p value < 0.016? (<0.05/3 biomarkers)
Thank you for anyone's help. 
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Yes, for Bonferroni correction you did correct. Basically, here are 2 ways of doing it and both lead to the same result: one way, as you did: when deviding threshold level (0.05) by number of tests. And the second way: to multiply obtained p-values for each test by the number of tests and if the adjusted p-value is still below 0.05, consider as significant. This is about Bonferroni correction.
But now, before doing the Bonferroni correction, clarify for yourself: did you do multiple logistic regression of univariate? If you did multiple logistic regression, you do not need any further correction. As I understood, you combined them, meaning that you did multivariate logistic regressoin analysis. So, just use the p-value you obtained. You could apply the Bonferroni correction to the initial analysis, when all biomarker were analysed between normal and cancer serum.
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Hi there,
we are focused on impression cytology of conjunctiva. We would like to isolate the cells from imprints on strips of Millipore filter membranes. We´ve tried mechanical isolation/detachement into the DMEM with microcentrifuge tube shaker and then vortexing. However the yield and cell condition are not good. Simply, the most of the cells are damaged. Does anyone have some experience with the cell isolation from filter membranes? Any hint can be helpful. Thanks a lot!
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In the publication attached, they use laser microdissection (MMI CellCut) to isolate cells from ISET filters. I hope this helps.
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I am studying siphonophore tentacle batteries across many species, using different microscopy techniques. I think I might have found the largest nematocysts known for all Cnidaria, as far as my literature search goes, but maybe someone has found something larger. Also, let me know if you heard of any intracellular structure larger than nematocysts.
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Thanks Joan! The ones I've measured are way larger than those, heteronemes >270um capsule length, >1M um3. I don't have discharge tubule length values for them yet, but it would be interesting to record that too.
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I've noticed several contributors on here implicating that ATP synthase could operate outside on the mitochondria. Under Peter Mitchels chemosomotic theory it is possible that the membrane potential created by calcium ion concentrations in endoplasmic reticulums could drive hydrogen ions down ATP synthase if present in the ERs membrane. I've spent sometime thinking how this could be demonstrated, unfortunately I have fallen short as the majority of assays i'm aware of use false substrates for the citric acid cycle to demonstrate ATP synthesis..
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Sorry , Idont no
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I would like to perform a immunocytochemistry (ICC) on differentiated THP-1 to evaluate the expression of a membrane receptor. I will use for that a primary conjugated antibody. On blocking steps do I really need to use a Fc receptor blocking solution or can I use regular blocking agents (like FBS or BSA)?
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In our experience we have saw binding of conjugated antibodies to macrophages only once. So technically it could be possible. Since serum contains immunoglobulins (in contrast to BSA) thus using of BSA or pooled human heat inactivated AB serum could be used as source of a concurrent ligands for Fc receptor and effectively downgrades false positive signal (if exist) to minimum.
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1. To germinate the seeds on petriplate and use the meristems of the root and dip into the nanoparticles solution for 0,1, 2 hrs ?
Or
2.First dip the seeds in nanoparticles solution for desired period and then allow it to germinate and then fix the root meristems for cytology assay?
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Dear Melissa Chernick,
Thanks for adding the answer along with reference papers.
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Does anyone have experience with Merk's Neoclear and Neomount solutions? Does this require additional steps after the standard Papanicolaou staining procedure?
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Tibor, just asking about your standard PAP-staining: which one do you use (when reading:
with 3 procedures: 
i)    Procedure 1 (Standard Method)
ii)   Procedure 2 (Modified Pap Procedure)    and 
iii)  Procedure 3 (Rapid Economic, Acetic Acid, Papanicolaou Stain Method)
I am wondering why you did not get any answer since 2012! (but admit that I found your question some minute ago....)....would recommend, to edit your question and add some keywords more: e.g.: Histology, Histochemistry, smears, Staining   to eventually increase readership.... best wishes and good luck...W.M.
NB: I haven't done PAP-staining during my former professional career...but perhaps I could help -at least knowing the protocol of your PAP-stain....
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I am veterinary pathologist and I am working on Pneumocystis in pigs. We want to establish a method and would need unstained slides from Pneumocystis jirovecii positive AND negative humans. Most publications deal with PCR or cytology, therefore it is really difficult to find somebody who may have formalin fixed paraffin embedded material. I really hope that there is any possibility to get material by this way. Thank you!
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Dear Ivan,
we will use the native samples. A method has been described for human Pneumocystis and we will try to adapt the protocol.
Greatings back from my colleagues!
Christiane
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Hi all,
I would have to irradiate lymphoma cell lines (Bl41 and J3D) and planning on looking for DNA damage clearance by IF following different nuclear markers. The center where I will irradiate the cells has no cytospin and so for short time point I would not be able to get the cells to my lab on time.
One solution would be to fix the cells before cytospin but I cannot find any protocol for that. Has anybody try to fix floating cells before cytospin?  
Thanks,
Matt
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No issue at all, I developed techniques over 20 years ago. Simply re-suspend your cells in medium and add formaldehyde (1:10 producing a 4% solution of formalin). I recommend using 'charged' slides for your cytospins and I spin at 500rpm for 10 minutes. The remaining cell suspension can quite happily be stored in the fridge for 12 months+ and more cytospins made at any point. I've used this for HCC, breast cancer, prostate cancer cell lines to name but a few. I've attached images of EBV+ lymphoma cell lines to demonstrate the results.
Formalin fixation obviously will then require some form of antigen retrieval technique, pre-immunostaining. Having been at the forefront in developing these in the UK in the early 90's I actually published on the use of microwave antigen retrieval for cytological preparations (Reynolds et al Cytopathology 1994; 5: 345-358).
Some say this is problematic for fluorescent techniques, but that is a myth. I use pretty much the same protocols for light or fluorescent microscopy, particularly on formalin fixed paraffin embedded tissues, as evident by my numerous publications showing this, eg Reynolds et al Hepatology. 2008;47(2):418-27.
Hope this resolves your problem and please feel free to contact me direct if you experience any specific issues.
Regards
Gary
 
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Hi all,
Here I attach the snapshot of the imaging area. These are RBC's forming clusters which affects my further calculations/experiment. How do I avoid this? Any coating to the channel would help? Thanks in advance! 
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Try to wash flow-chamber  (before measurements) by buffer that supplemented with 0.5-1.0 % of albumin.
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Cytologist, wheat, mitosis   
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Hi, Nader,
Cytogenetic plant (wheat) techniques, namely, Fixation, Pretreatment, and Staining, please, see at site
Godspeed
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I have always worked with whole tissues. Now I will work with cells. Granulosa cells will be obtained form follicular fluid (from follicle aspiration) after centrifugation. As I have never worked with these samples, I´m looking for a protocol to collect them in a slide, fix them, preserve them for a month and then perform an immunocytochemistry. thanks!
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You can make a cell block the technique you can found everywhere. 
But in my experience, instead of using commercial gel (ex Histogel), we use Agar gel 3%, it works well. 
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Hi,
I'm trying to analyze the ecological variation in two cytotypes of the same plant species. I do not have environmental data collected myself. I would like to use open source data such as "worldClime". I would like to see if there is a difference in the distribution of the two cytotypes in terms of environmental factors. I use R for my analysis. I would much appreciate suggestions about appropriate methods.
Thanks
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Dear Piyal
Though I never work on the ecological variation analysis methodology, but as a researcher on plant taxonomy I can guess how tough your work is.  actually for any sort of ecological variation analysis or study your own data about the climatic condition of the study area is needed, other wise if you depends on the data available in the internet system may distract your experiment due those data are not accurate in-every sense.  
besides these during analysis of the cytological variation it is also very important to take a grave look on the environmental condition because the expression of the gen often depends on various environmental condition. 
with best wishes
Sunit Mitra 
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Attached I send two documents in PDF with photographs of the sample, stained with MGG, another with Pap. Final magnification of 400x.
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these cells are the size of lymphocytes with very dense nuclei, I think  they could be apoptotic cells, perhaps due to therapy?
you can try confirm apoptotic cells by immunocytochemical detection of c-caspase 3?
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I use rat ant-mouse CD8a Ab(from eBioscience) to stain mouse pancreas and the Ab always stains the cells of secretory acini which looks like "specific binding", the "specific binding" not in islet. The CD8 antibody works well for mouse spleen,thymus, lymph node. I think it's pancreas tissue problem,  no matter how I try , like bake slides,  deparaffin, antigen retrieval, block, Ab incubation time and temperature, wash….the secretory acini staining always there, some area strong, some weak, no particular pattern(I'm sure it's not CD8 antigen). How can I get rid of the "specific" non specific binding? Thanks.
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Hi Maria;
Thank you very much. Your answer is very helpful and I'm going to use the 2 reagents for CD8 staining. So far for pancreas IHC , only CD8 has the problem, I did other IHC with primary Abs from rat and from rabbit, no such problem.
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Can anybody help me to get the protocol for wright staining? I want to stain the different cell present in sputum. 
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To Hamid:
despite the request he posted long time ago I would like to add here for the convenience of others:
go to the Reply from  Tam Quyet Nguyen (this thread, # 08) and you'll find the Sigma-Aldrich protocol....
best wishes and regards, Wolfgang
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I am studying 'chromosomal count' and 'mitosis' in tree plant. I have germinated the seeds in petri plate and want to study allelopathic effect of plant extract on cell division. Kindly help me in root tip treatment and staining technique for better results.
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sorry I am not a botany is not my speciality K.
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I am interested in acquiring a Doncaster counting dish to count AM fungal spores since the use of the Burker-Turk or Neubauer chamber is not suitable for this. Many spores are bigger than 100 µm so they get stucked between the two glasses. Counting directly in a petri dish is not easy since you don't have a path to follow and is easy to get disoriented. Thus, the Doncaster chamber seems to be the best option but I cannot find where to buy it. Any advice about providers or alternatives? Thanks in advance.
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Nosotros las conseguimos con colegas venezolanos del Instituto Venezolano de Investigaciones Científicas (IVIC), por favor explora estos contactos: Dra. Milagros Lovera: mlovera@ivic.gob.ve y Dra. Alicia Cáceres:alicia2001@gmail.com. Suerte con esta gestión, saludos cordiales de Eduardo
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In 1869, the resident physician at Melbourne hospital, Australia, Thomas Ashworth for the first time observed cells from a postmortem blood sample, from a patient who had about 30 subcutaneous tumors, that were morphologically identical with those of the cancer itself. This observation prompted him to suggest they “may tend to throw some light upon the mode of origin of multiple tumours existing in the same person”.
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You don't happen to have the publication available somewhere ... and a photo of Thomas Ashworth?
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I am looking for some databases to apply some techniques for the detection and classification of abnormalities in images of cytological tests.
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I am dealing with invertebrate tissues but my supervisor ifor bowen has a lot of papers about cancers you can search for i.d. bowen and also cathrin saraf
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Has anyone ever made their own PTFE-coated microscope slides? The ready-made PTFE-coated microscope slides are sold at a high price in my country, so I am wondering if it is more cost-effective if I simply buy a PTFE tape, and stick it to the available glass slides (we have drawers of glass slides in stock at the moment). What do you think?
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You could use a silane such as 1H,1H,2H,2H-perfluorodecyltrichlorosilane, commonly abbreviated as PFDTS.
It is rather inexpensive and I think it would do the job.
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Hey!
I just started working with NIH3T3 Cells and this week I conducted my second plasmid DNA transfection with lipofectamine2000 in this cell line.
Unlike the first time, which worked perfectly, 3 hours into the transfection I noticed my cells started dying and the survivors nuclei started looking like crescents or really thin donuts or onion rings and more like epithelial cells rather than fibroblasts. The transfection didn't work.
I work a lot with SN4741 cells and I used my SN4741 cell media to culture the 3T3 cells as all the others in my group are doing the same with no deleterious effects on the cells.
The main difference between our SN media and the 3T3 media recommended by ATCC is the serum (we use FBS and not BCS) and we supplement the media with extra L-Glutamine and Glucose.
Can someone tell me if this morphology is to be expected or if it is otherwise abnormal?
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Hi,
Maybe you should also take care of the quality/purity of your plasmid DNA, e. g.  by using an endotoxin-free plasmid kit when preparation from bacteria. The amount of DNA might also be a critical factor. Also, try out transfection without serum, if you did not so. Good luck.
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Hello everyone,
Is there any solution to reduce considerably the 4T1 mammospheres aggregation.Is there any other agent that can replace methyl cellulose? 
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Please see these papers. They may help you out.
Cancer Letters vol.323(1) 2012 20-28.
Good Luck!
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This is a picture about IHC result. We found that the protein was highly expressed in the nuclear and cytoplasm of spermatocyte, round  spermatids, spematogonia and Sertoli cells,  but was  hardly detectable in elongated  spermatozoon. I am confuesed how to distinguish second spermatocytes and round spermatids.
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I agree with Laura and Shahzad, it is not easy to detect secondary spermatocytes, specially when tubular sections are not completely obliques (since maturation stages are mixed). Performing complementary histological sections with PAS staining  could help you to differentiate stage XII, and thus secondary spermatocytes. But tubules must be completely rounded and with the lumen visible. You can look up my last work in which I combine immunohistological techniches with PAS staining in mouse testis.
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After a classical Ficoll, I spread PBMC (suspended in PBS1X) on Poly-L-Lysin pre-treated and positively charged glass slides with cytospin (200rpm during 5 min). Then I fix them with PFA 4% and cold methanol 100% before immunofluorescence.
Their nuclei look like those of polymorphonuclear cells or are broken (with "streaming DAPI").
Is this distortion known with cytospin (even so I reduced the speed and time of cytospin) ? 
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Try to fix with only 100% of methanol.  PFA (fixative) consist sodium and formic acid which usually causes some shrinkage and/or distortion of the specimen. This might caused the distortion of the nucleus. Most of the staining (e.g: Wright's staining) for thin blood smear (obtained using blood smear or cytospin) requires methanol as the fixative agent.
All the best.
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What are the differences between cytology, cytogenetics, and karyology?
How do they deal with each other?
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Agreed with Andrew - apart from the fact that cytogenetics covers whole field of chromosome research - not only in human, but also animal, plant, fungi...
Also male human karyotype reads as 46,XY according to ISCN(2013).
As soon as you do molecular cytogenetics in interphase nuclei you also may say here you start to enter the field karyology.
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Hello
I have a really confusing question. I wanna count the cell of liquid based cytology(LBC). But the cell morphology is different between normal LBC and cancer LBC. And normally, cancer LBC contains so many sediments which with red color. I have no idea that if the red sediments also are cells or may be other debris. 
Could anyone help me?
Thank you!
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Cell counting depends on the type of cells you require.If the sample contain only one type of cells,you can use an image analysis software easily.Otherwise you have to train the software first.
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I have read Levan et al. 1964 and Atlas of Mammalian Chromosomes, but I am still want to know proper nomenclatur of mammalian chromosome pair number, especially in bats and rats. Thank you so much
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Thank you so much Prof. DC Gautam
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Cervical cytology images contain nuclei,cytoplasm and background. If the nuclei overlap each other what method can be used for segmentation
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There are different methods for touching nuclei seperation in literature. Before segmentation find out the seed points accurately. Euclidian distance map with multy scale laplacian of gaussian, radial symmetry transform,single path voting with shifted gaussian and concavity point detection are some of the technique seen in the literature. I also need matlab code for these. if any one has the code,pls help  
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I found a publication about chromatid break in mice. The mice were induced by some chemical subtances. So, in the natural condition (wild) what's factor that makes chromatid break happen?. Thank you ^^
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There are several factors causing chromatid breaks in natural conditions: i) replication across a nick would give rise to chromatid breaks; ii) a second cause is oxidation from radicals occurring during normal oxidative respiration; iii) failures of DNA  topoisomerases; and  iv) natural ionizing radiation including gamma rays and X-rays.
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I have conducted karyotype of bone marrow in the wild
But, I have never try karyotype using blood. I need some methods that really useful and work. I think blood is more easily contaminated, so it must quite difficult
Thanks
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Sounds good thank you so much for both of you Sonal and Khaled :D
I have to try it
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Dear Researchers, I have been having difficulties in determining the chromosome numbers of some accessions of Hibiscus sabdariffa germplasm in Nigeria. Common cytological procedures for both mitosis and meiosis had ben used, yet no result. What should I do?
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@Please enlist the difficulties/observations you have come across.
Regards
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When red blood cells are stimulated by toxic compounds (e.g. mercury) they change morphology to acanthocytes. Most part of papers report SEM analysis; is it possible to apply a flow cytometric method?
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In theory, you might be able to identify your acanthocyte population simply by using Forward and Side scatter, as these cells appear to be smaller and possibly more granular than typical erythrocytes. Have you ever looked at your control and toxin treated cells via SSC/FSC? Happy to have a look, if you could post a picture of your flow cytometry data.
AnnexinV is used to detect phophatidylserine exposure - this is for example the case f