Mahendra Singh added an answer:I want to use Histochemistry for detection of protein and lipids. Can anyone suggest a procedure without using Cryocut?
I had all basic instruments regarding histology
Several protocols are available for histochemistry without cryostat section
but always keep in mind that if you are using the samples fixed very long back may encounter problems with antigen retrieval.
So better to give minimum fixation time.
What kind antigen you want to locate in birds.
If you need any help you can contact me @ firstname.lastname@example.orgFollowing
Belal Hassanzadeh added an answer:What is the best stereological method for quantifying cellular density?
I am looking for an efficient method of quantifying the cellular density of murine hippocampi. I have already imaged my dapi-stained tissue. Using ImageJ, I have tried adjusting the threshold range and using the ITCN cell-counting plugin, but did not get accurate results. Has cavalieri point-counting shown to be efficient for cell counting? Any ideas would be greatly appreciated.
To my mind you have to estimate the total volume of nuclei [using the cavalieri method] then calculate average volume of the nuclei [average of about 100 nuclei]. Finally total n.V./avrage n.V.=total cell number , now the cellular density= total cell number/volume of hippocampi
Vuong Tran added an answer:Does anyone know a histological image analysis program?
I'm looking for an image analysis program in which I can make some measurement in histologic sections of cartilage. I am especially interested in a program which will make it possible to give surface area information on a manually selected area. Also drawing lines, to divide it into three equal parts would be nice. Does anyone know such a program? Photoshop doesn't have these tools unfortunately.
ImageJ is my recommendation, as it is a freeware and easy to use.
Photoshop also has the counting and measure tool. I often count the cell by photoshop because I'm more familiar with it.Following
Wolfgang H. Muss added an answer:How do I do a liver cryosection oil red o staining?
I am trying to do oil red o staining of liver cryosection.
I can not see any lipid droplet from the section after staining.
I used oil red o solution from sigma, but nothing was stained.
The thickness of the section is 5 um.
I performed 3 different general protocols.
1) propylene glycol method
2) 2-propanol & DW method
3) only oil red o solution and washing with 85% propylene glycol
If you guys have any suggestion, please help me out.
Dear Jeong Hoon Pan ,
don't want to interfere with all the replies you got already. I could add perhaps other recipes or an opinion on your "failure of staining". I just would like to point also to another - but very short - thread on RG: https://www.researchgate.net/post/Can_anybody_suggest_the_best_method_for_oil_red-o_staining_of_differentiated_adipocyte_cells_derived_from_Whartons_jelly_stem_cells#view=54de0e1fd039b18b7a8b4690 .
But if you would like to browse a bit the "matter" of Oil Red O-staining contained in RG, you should go to:
(it is the <search function> you can use in the field right to JOBS find the sketch for "person" which makes you able to search for researchers names): find SEARCH, pulling down the menue, choose "QUESTIONS" and insert your search phrase/Key words, in this case: <Oil red O>).
The results are a lot of valuable questions and answers....
Best wishes and good luck
Daniil Rotin added an answer:Does anyone know how to reliably differentiate between adrenal adenoma from metastatic RCC in adrenal gland?
IHC, EM or simple histology - everything is OK, but I need 100% of assurance.
I always mean HISTOPATHOLOGICAL level. In addition, is anybody able to distinguish between those entities on Frozen Sections?Following
Can anyone share a protocol for mouse pancreas processing and embedding?
I need to do histochemical staining of mouse pancreatic tissue. I tried few protocols but the tissue came out dry and very friable during sectioning. I searched online and couldn't find specific protocol for pancreas.
Francisco: Tissue, that was not properly dehydrated and penetrated by paraffin looks totally different from over-deyhdrated tissue. And the problems while cutting are also very different.
Kiernan refers to insufficient dehydration and the remainance of organic solvents in the block. The effect while cutting will be, that the block-surface is smeary and wet, also white sometimes. And the section will "explode" on the water-surface.
Over-dehydrated tissue is hard, brittle, remembers sometimes of "wood". While cutting the surface turns rough and white. The section keeps its form, but shows holes and streams.
So bad processing may lead to different outcomes due to different causes.Following
Evelyn H Schlenker added an answer:Does anyone know the correct way to cut the lung sample for asthmatic mice?I would like to see the effect of certain drug on the lung inflammation of ovalbumin induced mice however I am not sure how to cut the sample for H&E and periodic acid schiff (PAS) staining. Is it longitudinal or cross section? Can anyone help?
I also like longitudinal slices through the left lobe to evaluate histopathology of the mouse lung. You can see the distribution of abnormalities best this way.Following
Waleed H Omer added an answer:How can I improve the tissue integrity of frozen samples in cryosectioning?
I am trying to isolate specific cells from mouse testis with laser micro-dissection and am preparing tissue sections to locate my target cells. As my final objective is to get the RNA, I avoided fixing the tissue before the cryo-section and when I checked the tissue under microscope after Hematoxylin staining, I found it hard to identify most of the tissue's details. I also repeated the experiment using Ethanol and Methanol as fixatives, but that also didn't help to solve the problem. To be mentioned here is my samples are frozen and I can not get fresh ones so I have to use it this way.
My question now is how can I improve the tissue integrity of my sections? I appreciate if you can give me any ideas on that.
Thank you all. Wesley I think you made a good point that I should consider. I will give it a try.Following
Andrew Collins added an answer:Why are my spinal cord sections curled after IHCs?
So I'm working with mouse spinal cords and sometimes the sections are curled/rolled after being sectioned/IHC. It doesn't happen to every batch processed but a good portion do. What would cause that? The tight curling makes it extremely difficult to slide mount. Is it the perfusion? sectioning? iHC protocol? I don't know where to begin troubleshooting.
Here is my current protocol:
The animals are perfused and fixed using 4% para, the lumbar region is removed and placed in a vial of 4% para for one hour. Athens cords are then transferred to a 30% sucrose solution for 3 days then sectioned on a Lecia cryostat at 40micrometers. Sections are placed in cryoprotectant for short term storage for IHCs.
Any assistance would be much appreciated.
I use a couple of small paintbrushes. When the section is cut, I flip the section over so that the fold faces towards the slide. As you place the slide down on top, the section should naturally flatten itself out, without you have to apply much pressure.Following
Smita Bhutda added an answer:IS there a staining for adherent and tight junction protein in fixed cells?
I have immortalized Human Brain Endothelial cells. I am trying to stain VE-Cadherin and Occludin in this PFA fixed cells. Instead of getting staining at cell periphery (On cell membrane), I am getting staining in the cytoplasm. I had tried different fixatives like PFA and methanol, with and without permeabilization, permeabilization with different concentration of triton x 100 ranging from 0.05% to 1% PFA. Please have a look to the attached image.
Can anyone suggest me something to get staining on cell membrane.
I agree with you Jean-Martin Lapointe. But the staining is not looking like membrane staining even in z Stack mode of microscopy. Also people have used same cells to get junction staining for occludin and they have got nice peripheral ring structure. I did follow exact same protocol as them but I did not get it. I wrote them but I think they were too busy to reply.
This time I will overgrow cells and during imaging I will use z-Stack mode.Following
Giulio Cecchini added an answer:Is there any difference in histological appearance between glioblastoma at first surgery and at recurrence?
If you compare the histological appearance of glioblastoma specimens from the first resection and from recurrence, is there any difference? If so, are there any publications describing this?
When talking about low grade gliomas, indeed, the recurrence shows a different histopathology in the majority of cases.Following
Flavie Sicard added an answer:What histology studies exist of the adrenal cortex in individuals specifically and only with low 'normal' serum DHEA levels?
The so called normal range for serum DHEAS and DHEA are unproven, and especially so for levels in the lower part of this range. On what basis has this range been defined, and what does the adrenal cortex look like in individuals or animals who have constitutive or pathological alterations/ reductions of these serum levels of DHEA?
You can find quite good histological studies in human adrenal describing changes in DHEA/DHEAS related to aging or adrenache in:
-Dharia et al., Effects of Aging on Cytochrome B5 Expression in the Human Adrenal Gland, JCEM, 2005
-Hui et al., Development of the human adrenal zona reticularis: morphometric and immunohistochemical studies from birth to adolescence, JOE, 2009
-Gell et al., Adrenarche Results from Development of a 3β-Hydroxysteroid Dehydrogenase-Deficient Adrenal Reticularis, JCEM, 1998
You should also find numerous reviews from Pr PJ Hornsby or Pr WE Rainey about this subject.Following
Dorothy hu added an answer:Why during sectioning of undecalcified plastic embedded bone, sections start to tear in region of secondary spongiosa?
I would highly appreciate if someone could give me an advice regarding plastic embedding. I am working with the bone tissues, and I am trying to establish embedding process in Spurr resin in order to analyse dynamic histomorphometry. Bones are from 6 months old mice. First, I dehydrate them in 70%, 95%, 100% ethanol and 100% acetone (every step takes 2 days) and then start infiltration process with Spurr resin 50%, 75% (mixed with acetone) and 100% (also steps take 2 days). Dehydration and infiltration processes take place in desiccator connected to vacuum pump. At the end of infiltration, bones are embedded in 100% Spurr and plastic blocks polymerize at 55oC for 2 days. When I do sectioning, I have this problem that region of secondary spongiosa starts to tear, and also block by itself is very brittle. What could cause this problem, could it be that infiltration was poor, or the ratio of Spurr components are not good?
Thank you for any answer and help,
Paraffin bone tissue has to be decalcified. It can not do dynamic histomorphometry. Try
Jirko and others suggestions. They cover pretty much all you need to do. We use MMA mixture and only dehydrate for total three hours from 90% to 100% acetone before infiltration step mouse long boneFollowing
Regina Vontell added an answer:Any suggestions on Alcian blue/nuclear fast red counterstain on paraffin slides?
I'm going to try an alcian blue/nuclear fast red staining on heart mouse paraffin slides. All the protocols that I've found in internet provide a 30' of incubation in alcian blue. Is this time enough? Is there anybody that can share with me some tricks about this staining protocol? The paraffin section that I'm going to use are very precoious.
I also recommend Gudrun Lang, advice and take care when using the nuclear fast red. The dehydration steps may dissolve some of the staining, if so you can always rehydrate and repeat it.
Can you get some samples of intestine to run as a control? There are a few companies such as, Newcomer Supply that sells control tissue.Following
Marcia Gasis added an answer:Can ice-damage be completely avoided when embedding frozen brain tissue and how?
Recently we are desperate about ice-injury of mouse brain slice in HE staining. There are so many small holes in our 8um slice, probably caused by ice-formation. We perfused our mice and used 20%(mass ratio, solute/solvent) sucrose to dehydrate their brains, still a lot of holes as a result. At freezing step, dehydrated brains were transferred immediately into -80 centigrade fridge, maybe it was not quick enough?
But some published paper also display photo with similar injury, so can this be fully avoided and how to mitigate as much as possible?
Thanks in advance for any advice!
Looks good! The small "holes" are blood vessels.
Ayman EL-Meghawry EL-Kenawy added an answer:Can H&E staining be used on in-vitro samples?
I am looking for a staining method to detect apoptotic cells vs. necrotics using bright-field microscope (in fact for an easy, quick, and cheap assay!). I know H&E is used for tissue, I would like to know if it can be used for cultured cells too?
Giemsa staining is the most appropriate staining for what you asked.
Carol Whitinger added an answer:Does anyone have a protocol for tendon cryosection?
I'm recently working on mouse tendon cryosection for immunostaining. But when I cut these tendon samples, they scattered so it's very hard to get intact sections. I've tried to changed the cryostat temperature up or down, and also tried snap freezing fresh samples or 4% PFA fixed and 30% dehydrated samples. Does anyone have experience with tendon cryosection?
Sometimes thinner sections have less chatter than thicker sections; try sectioning at 8 microns. Also, have you explored any tape transfer techniques?Following
Does anyone have a protocol for double chromogenic staining using DAB and AEC on paraffin-embedded tissue?
I have one that works well for DAB using biotinylated 2°Ab, Strep-HRP and unconjugated Tyramide. (I use DAB pellet from Sigma, diulted in Tris-HCL solution).
We have as well HRP 2°Ab but nothing for AEC detection. I saw many different protocol and products for AEC staining but never one with all details for this specific double staining.
Thanks in advance for your help!
I never heard, that someone used alkaline phosphatase for developing AEC. For double-staining: first IHC-part with peroxidase and DAB, second IHC-part with alkaline phosphatase and eg. BCIP-NBT or permanent red http://www.dako.com/at/ar38/p224598/prod_products.htFollowing
Mohamed Najimi added an answer:When analyzing images of fibers in coronally-sectioned mouse brain, is it possible to differentiate between fibers of passage and terminal fibers?
Does anyone have a method to determine the difference by morphology alone i.e. without the use of an additional label such as synapsin for terminals or tau for axons? Our neurons are filled with virally-expressed eYFP.
By classical IHC (using peroxidase for example), you can distinguish between fibers of passage and terminals. These latter are generally presented as varicosities, but this does not mean necessary that they correspond systematically to terminals. Indeed, depending on sectioning plans. Fibers of passage are presented as continued filaments with different sizes. In summary, you don't need specific method nor specific antibodies to visualize them.Following
Warren M Meyers added an answer:Does anyone have a good protocol to reduce/remove autofluorescence in tissues?I need to performe some immunofluorescent assays in embryonic mouse lung paraffin sections, fixed in formalin. However, the autofluorescent signal (both green and red channels) in the negative control is a bit high, so I cannot really validate my results. How can I solve this problem?
Thank you Nagar, that is greatly appreciated. Sincerely, Warren.Following
Martin Bertz added an answer:What are these structures in my liver samples?
a colleague of mine was recently baffled when looking at liver tissue coming from IL10 knockout mice which spontaneously develop a chronic inflammatory bowel disease.
Can anyone with experience in histo(patho)logy tell me what the brighter structures in the attached picture are? Is this some kind of fibrosis or what are these cells?
Thank you alot in advance!
Hi Robert, thanks for the insight.
As far as I know the tissue was directly "bathed" in 4% formalin until further processing (standard embedding in paraffin). However, it was only this particular animal showing these structures and not any other littermate. I guess if there was a problem with fixation we would see it in other specimen too...Following
Madhukar Baburao Deshmukh added an answer:Can Xylene act as a substrate for the second part of the mannich reaction during formalin fixation?Under the mannich reaction, after the initial amine-formaldehyde reaction, could xylene act as a substrate to stabilize the reaction if the reaction does not stabilize during or prior to dehydration and clearing? In synthetic chemistry, amine-formaldehyde reaction products can attack ring structures, is this possible under the FFPE processing conditions?
Mannich reaction during formalin fixation you need a substrate having active methylene group like actopphenone & not xylene.Following
Nathan Ahlgrim added an answer:How can I label brain hemispheres for free-floating histology?
I am running immunohistochemistry on rat brain tissue, and the sections will be stained while free-floating. However, we need to reliably know the left from right hemisphere. We cannot take a notch out of the cortex (which we have done previously), because we are taking a full survey of the brain.
Could someone recommend a method to label the hemispheres? Is there some sort of marker or pen that is visible to the eye but would not affect staining?
- Thank you -
Thank you all for your advice.Following
Michel Paquet added an answer:How would you stain a one centimeter section of fresh sciatic nerve for cell viability?
LIVE/DEAD stain : AM stain : LDH : Post-fix staining
Perineurium is a diffusion barrier
Thanks for your kind help.Following
Maximiliaan Lens added an answer:Why does my Goldner stain fail to stain metaphyseal mineralized bone?
I have performed a goldner trichrome stain to perform histomorphometric analysis of the metaphyseal zone of MMA embedded bone. But I fail to stain the mineralized metaphyseal bone, the rest of my bone sample isn't stained very clearly as well. I followed the correct protocol and looked at amendements, but can't find the problem. Can someone provide me with some help? Thanks in advance.
(other pictures can be provided on demand)
my goal is to use the Goldberg stain for some quantitative measurements on bone samples from top studies. But as you see, my metaphyseal zone is not colored appropriately, so I can not really measure anything.
my email is Maximiliaan.email@example.com
thank you for your time and sorry for the late answer,
How long should a tissue submitted for histology remain in neutral buffered formalin?
Some tissues might be overfixed, can this affect the outcome incase you need to perfome immunohistochemistry e.g. for lymphomas.
Rose: a question about the brittleness of the mouse-brains.
When you touch the mouse-brain after fixation, do they feel really hard or brittle?
In my experience brittle outcome of tissue is more a matter of over-processing. Too long dehydration, too long xylene, That leads to over-dehydration and de-fatting of tissue. For sectioning this can partly be emproved by soaking the trimmed paraffin-block in water for a few minutes.Following
Jeanne Pawitan added an answer:Does interstitial growth of cartilage give rise to isogenic groups of chondrocytes as a final aim?
Or it´s only transitional stage of chondrocytes formation which is separated into a individual cell of chondrocyte later? And why should it separate, whereas it differentiates from a single chondroblast? Couldn´t it just differentiate from a single chondroblast?
I mean, if it´s give rise to isogenic group, what´s the purpose of this formation? What it benefits? Why not stay still in form of an individual cell?
Sorry to keep asking such basic stuffs that an undergraduate should´ve already known well.
Dear @ Daniels,
Thanks for the correction. Yes, it is stiff, not hard, because bone is hard.Following
The study of the microscopic anatomy of cells and tissues of plants and animals, including tissue fixing, fixation and staining.