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Liver - Science topic

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I have a problem concerning my liver fluorescence staining: one primary antibody, GARP, in the attached picture in red, is causing these weird stainings on and around the nucleus. Does anybody happen to know how to improve my staining and get rid of these false positive stains?
Or general advice for liver staining, as the autofluorescence in liver tissue is always really high.
We are cooking our samples in citrate ph6 buffer, and using Avidin+Biotin as well as Tris,TWEEN20 + 5% BSA for blocking. In order to attach the fluorescence dye we are using Dylight 550.
I would very much appreciate any advice!
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generally, use a negative control with only secondary antibody in the whole staining process to ensure that this is actually a false positive stainings.
did you do this? furthermore, try to use different concentrations (dilutions of primary and secondary a.b.). that migght help too.
ensure that you use blocking solution and serum of respected host of primary a.b. to block.
lemme know!
silke
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The US CDC knew very early, from post-marketing Adverse Reaction reports, that the COVID-19 vaccines were causing numerous Amyloid fibril diseases including Amyloidosis subcategories: Cerebral angiopathy, Senile, Cardiac, Cutaneous, Dialysis, Hepatic, Pulmonary and Renal.
Elegant work by Swedish researchers demonstrated that Synthetic Spike Protein can be broken down in vitro by incubation with the protease Neutrophil Elastase into short peptide sequences that can induce damaging amyloid-like fibrils.
References: Vaccine Adverse Event Reporting System (VAERS) Standard Operating Procedures for COVID-19 (as of 29 January 2021)
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Recent article on Amyloidosis caused by Spike Protein
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Coronavirus disease (COVID-19) pandemic is a global health problem. Infected patients usually have respiratory symptoms due to lung involvements. However, liver impairments could be another findings.
So does Covid-19 affect liver functions & how?
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Hello everyone,
can somebody recommend me an fgf21 Elisa kit which actually works on tissues such as skeletal muscle or liver?
I ve found a lot of kits that has tested on serum with good results, but I am not sure about their efficiency on tissues.
Thank you in advance.
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Cusabio has FGF21 ELISA kit for human, mouse, rat and bovine specifically. If you want to test tissue samples, it is recommended to do a pretest first. You could search "FGF21 ELISA" on the website, https://www.cusabio.com/, to view more details.
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In partial hepatectomy models, fat accumulation occurs during hepatocyte regeneration. Whether it is needed is controversial. Though it was initially thought to be the "fuel", later experiments have shown that proliferation can proceed even if fat accumulation is disrupted. My question is, what triggers this accumulation and what is its function.
Thank you
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Hepatic lipid accumulation after partial hepatectomy is a consequence of enhanced fatty acid mobilization and metastasis to the liver, as well as increased hepatic lipogenesis and decreased secretion of very-low-density lipoprotein.
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Literature suggesting hepatitis / liver injury following COVID vaccination. A latest peer reviewed article suggests SARS- CoV -2 vaccination (Pfizer)can elicit CD8 T-cell dominant Hepatitis (Autoimmune hepatitis).Details attached in document .Should such rare side effect be taken into consideration during vaccination?
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COVID-19 vaccination can elicit a distinct T cell-dominant immune-mediated hepatitis
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I am going to use healthy liver cell line for the cytotoxicity experiments with nanomaterials. So, I am wondering that are there any differences between THLE-2 and THLE-3 cell line ?
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Hello !!!
I am working on the hepatoprotective effect of some medicinal plants and, i want to use the ccl4 induced acute liver injury ( 7days protocole)
there are a plenty of articles that treat the subject, and each one use differente concentration !!
so according to your experience what is the best and most used ccl4 concentration to induce mice acute liver injury ??
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We got the best resalts using 0.5 ml/kg CCl4 to induce fibrosis
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We made a pilot study with N=2 mice per group to test biodistribution of rhodamine 6G-loaded silica nanoparticles functionalized with PEG, at 1.5h, 6h and 24h. We used GFP detector in IVIS Spectrum 200 imaging system and normalized fluorescence efficiency to control organs average, as a ratio (N=2 as well).
We expected to find high accumulation in liver, spleen and lung, possibly decreasing with time, but surprisingly we cannot see any rhodamine signal in liver at any time when compared to control organ. We made sure the biliary vesicle was not present and liver was properly washed. Signal in spleen is not very high either. Nevertheless, lung signal at 1.5 hours is 5-fold the controls signal, decreasing in time and disappearing at 24h.
We know optical imaging is not the most sensitive technique to assess nanoparticle biodistribution, but we can clearly see rhodamine signal in lung. What could possibly be going on in liver and spleen? Could their color or opacity cause any quenching of the signal? I've found this article detecting rhodamine in liver using the same equipment as we do () so I guess it is not a matter of quenching...
Do we have the best and most biocompatible nanoparticles ever? I don't think so! But we need an explanation for the lack of liver uptake...
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Dear Rob Keller,
Thank you very much for your kind answer!, I did not realise there was such a difference!
Still, to avoid undesired interactions that could lead to different biodistributions (nanoparticles like ours are usually uptaken by RES organs), or dye degradation, in our case rhodamine is loaded *inside* the nanoparticle. Thus, it is not exposed to plasma, so I do not believe it is a matter of solubility (at least, not rhodamine-related)...
I'll keep on researching!
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I have been performing immunofluorescence staining for liver tissue that is embedded in paraffin. I have been getting inconsistent results and the full liver section never stains through properly. There are always parts of the section that don't stain at all, or stain very poorly.
I know that the antibodies work well. I'm not sure if there's an issue with paraffin removal or antigen retrieval? I heat the sections for at least 30 min at 60C before the xylene step. Then I do the following:
[2X] Xylene, 7 min
[2X] 100% EtOH, 5 min
[2X] 70% EtOH, 5 min
[3X] rinse in autoclaved water
Antigen retrieval in 1x DAKO Target Retrieval reagent in pressure cooker.
I am attaching an example picture. I would greatly appreciate any trouble shooting help!!
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This image is an example of HNF4a staining. It is supposed to mark hepatocytes which are present throughout the liver. I have considered frozen sections, but would still like to optimize the paraffin-embedded protocol, since it achieves better morphology.
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The characterization of heavy metals like gadolinium in organs like liver, brain, lungs can best and simply be done by.....................
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Thank you, your responses and effort are appreciated.
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Dear all,
Which is the best mounting medium for liver frozen sections stained with oil red O? An aqueous one as Ab104135 could be the solution (even if suggested for IF)? Thanks in advance.
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3. Glycerine Jelly Mounting Medium
Gelatin (Kitchen grade)    10 g       Distilled Water                60 ml
Glycerol                       70 ml Phenol                            0.25 g
Dissolve the gelatine in the distilled water using sufficient heat to melt the gelatine, add the glycerol and phenol. Mix well and transfer to a small capped bottle and refrigerate.
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I did a qpcr reaction with SYBR master mix, and cDNA from epididymal adipose tissue. The RNA used for cDNA synthesis was intact (two bands on the electrofluoresis gel), good A260/280 and A260/230 ratios. With the cDNA from adipose tissue it did not amplify none primer. I did it with a liver cDNA and it worked. The tested primers were from genes such as PRDM16, UCP1, Beta Actin. Not even with the one of Beta actin it amplified. Could it be some inhibitor present in RNA or cDNA? Any suggestion?
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How about your positive and negative control? Is that valid?
If Beta-actin also didn't amplified, it is either there is inhibitor in the master mix or samples or there is no cDNA inside the sample.
Beware of ethanol or alcohol, which can serve as the source of inhibition. If your positive also without amplification, it might be the problem of master mix or qPCR protocol.
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We have realized a toxicological experiment with Wistar rats. Liver was collected and fixed wtih formallin. Histopathological analysis was performed from HE stained sections. Now, I am looking for a more specific method for analysing liver lesions from these formallin-fixed material or embedded tissue. Unfortunately, I do not have molecular techniques nor immunohistochemistry. Is there an histochemical method or histological staining specific necrosis detection? Thanks for helping!
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While a pt. Use HMG-CoA reductase inhibitors , it cause increase in liver enzyme ( 3 times the normal) and stop HMG-CoA reductase inhibitors. then it expect that liver enzyme will go down & when it become normal can we reintroduce statin again?. And when to recheack liver function again? I mean after how many wk. Of use of statin recheck liver enzyme again?
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I have two chemotherapy drug (A and B), at baseline, after first cycle, second cycle and third cycle, I want to compare between two drugs after each cycle and and which drug associated with more adverse events with time at the end of third cycle and after each cycle?
Some safety data is continuous such as liver enzyme, and some is categorical such as grade of some adverse events.
I think for continuous data repeated measure ANOVA can help but what about categorical variables?
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It is dependent from data: parametric or nonparametric.
When we have parametric data, to be used: paired t test, unpaired t test, Pearson test, ANOVA.
Nonparametric tests are: Wilcoxon test, Mann-Whitney test, Spearman test, Kruskall Wallis tests.
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Hi all,
I have been working on optimizing lysis conditions to do whole proteome lysis from liver tissue and have a head-scratcher. Using the BioRad detergent compatible BCA analysis kit, I get a woefully low estimation of protein extracted when compared to doing a mass balance (weighing empty tube, liver, then remaining pellet after extraction)... I've washed the liver tissue as much as possible to remove blood (non-perfused at harvest) and the samples aren't bloody looking. Does anyone have any suggestions of expected protein extracted per wet liver weight? If I know that, I can at least have a better idea of which number I should use (BCA or mass balance).
many thanks
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As a very general rule of thumb, the protein yield after tissue extraction in SDS or urea buffers with sonication/bead beater for total cell lysis, including organelles (whole proteome extraction) will be approximately 5-10% of the initial wet weight. So from a piece of tissue that weighs 100 mg, one might expect 5-10 mg of protein. This varies of course depending on the type of tissue being processed, the stringency of the lysis/extraction reagents and process (are you pulverizing the frozen tissue first?), whether the tissue is dehydrated, and the volume of buffer used for lysis.
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protocol or any relevant article please share
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CN103237888A
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Hi,
I am starting to work on a liver histopathology image classification project and looking for publicly available datasets. Any guidance related to this will be really helpful for me.
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I think Ankush truth answer .
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Plaques form due to a self-healing mechanism of blood vessels and will increase over time. When entering blood vessels, they block blood flow, lead to hypertension and decrease blood flow to organs such as the heart. To get rid of these plaques, we need to boost the good cholesterol such as HDL or improve health of liver to produce enzymes that move these plaques. So, what other ways to get rid of these plaques without using invasive methods?
Thanks and best regards.
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The following RG link is also very useful:
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Currently I'm trying to adjust Oil Red O staining method for liver lipids. Base protocol is A. Mehlem's (Nature Protocols; Vol. 8, No. 6; 2013; p. 1149-1154). I tried different times of ORO staining and counterstaining with hematoxylin. Had some problems with non-specific staining of cracks between cells which might have been solved with varying times. As I believe in the pics the darker dots are lipids and red colour between cells is other type of lipids? Maybe someone who has experience with ORO staining method could explain if attached pictures looks good and if not maybe suggest some corrections which might improve staining?
Liver samples are from chickens treated with high concentration of TBT.
Sample 7: 5 min. in ORO, 15 s. HTX, 30 min. wash; S8: 5 min. ORO, 1 m. HTX, 60 min. wash; S12: 5 min. ORO, 3 min. HTX, 30 min wash; S13: 5 min. ORO, 1 min. HTX, 30 min. wash.
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Reagents need to be filtered (Oil Red O).
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I have two unique teleost species, Saffron Cod and Broad Whitefish. I am analyzing the HSP70 concentrations in the cranial, hepatic, and muscle tissues. The Broad Whitefish samples all worked perfectly. However, the liver and muscle tissue samples for the Saffron Cod aren’t running (the cranial tissue samples are working perfectly). The HSP70 protein seems to be aggregating and sitting on top of the well.
My advisor and I believe perhaps the cod have a higher concentration of fats and thus aren’t separating like the other samples. All of the tissue samples were prepared the same way using a homogenization buffer with 4% SDS and Tris-HCl. The tissue samples were homogenized in the buffer, boiled for 5 minutes and spun down at max RPM for 15 minutes before extracting the supernatant with the protein sample. The protein samples are placed in 2xSB and boiled for 3 minutes before being loaded in 10% gel. All of the solutions, both cod and whitefish, are clear and fluid when at room temperature (none are gelatinous or are behaving weirdly). I have attached images of the gel and Western Blot with four Broad Whitefish samples (BDWF) and four Saffron Cod samples (SFCD). Muscle tissue is MT, hepatic tissue is HT, and cranial tissue is CT. The Western Blot for the Saffron Cod muscle sample didn’t show up for some reason, but when I ran it the other day it looked exactly like the liver tissue; the band was sitting at the very top of the well. Thank you for any and all help!
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Can we combine alcoholic and non-alcoholic liver disease into a single fatty liver risk scoring system?
If no, why?
If yes, what will be the potential of such research and implications.?
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Hello,
I'm trying to find suitable antibodies to discriminate hepatocytes from other cell types in the mouse fetal liver. I was considering ASGR1, but I found that it can be expressed in monocytes as well, or ABCC2/MRP2, but I couldn't find a nice antibody (please help in case you know one).
Any good (fetal) hepatocytes marker would be good for me.
In an ideal world, I would prefer a conjugated antibody against a surface marker for flow cytometry of live cells, but also an unconjugated antibody for immunofluorescence (in this case it can be an intracellular marker). I want to confidently distinguish hepatocytes from other cell types such as endothelial and haematopoietic cells.
Many thanks!
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I would use a multiparameter approach as many markers are expressed by multiple cell types. Use TER119 and CD45 to identify and eliminate hematopoietic cells. Don't overlook autofluorescence and light scatter to help identify large cells, versus the many smaller cell types.
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Investigation of apoptosis in liver tissue
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You can evaluate apoptosis in liver cells by several ways. Its not clear under what experimental conditions you are evaluating apoptosis in liver cells. TUNEL Assay for DNA fragmentation and Cleaved caspase-3 analysis by WB are of course convincing markers for apoptosis. The DNA ladder by agarose gel electrophoresis is considered as hall mark feature of apoptosis.
Indeed, depending on the availability of chemicals, antibodies facilities, you can explore whether death receptor pathway (FasL, intrinsic pathway) or mitochondria-mediated (extrinsic pathway; ROS-mediated) or both are operated in liver cells under certain experimental conditions. You can also go for Bax/Bcl2 ratio, P53 expression and other upstream caspases7/9 by western blotting could strengthen your findings on apoptosis.
As I said above, there are several potential markers of apoptosis, however its up to you and facilities available in your lab that can enable you to do quality grade apoptosis research in liver cells (hepatocytes).
Good Luck!
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I am conducting experiments to repair the meniscus injury of rabbits by transplanting cells, and rabbits are also given daily injections of tacrolimus, an immunosuppressant.Now I want to test whether the transplanted cells have adverse effects on the liver and kidney functions of rabbits, but I don't know which liver and kidney items need to be tested.I hope friends who have had similar experience can give some suggestions.
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Thank you very much for your reply. Your answer is very meaningful to me!
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Can anyone help me choose primary antibodies for mouse liver hepatocytes and also in conjunction Cd8 cells. Also Cd3 cells. I want to look at hepatocytes and adjacent immune cells in mice with augmented malaria infections. Thanks. Patrick. I need antibodies that react to mice hepatocytes. And also mouse cd8 and Tcells.
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ممكن ذلك من خلال البحث عن احد يساعدك في ذلك
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This study was carried out in order to find out the level of sheep’s meat, liver and kidney contamination by heavy metals such as: copper, lead, zinc, cadmium and cobalt in different areas of al Sulaimanyah Governorate in comparison with international allowed levels. For the above purpose; three samples of (meat, liver and kidney) were taken in three different
districts of al Sulaimanyah Governorate were covered: Said Sadiq, Dokan district and sulaimanyah city center. The samples were collected during October and November 2020. The triple interference (factors) has affected significantly, the amount of copper in the different sheep tissues; so the amount was varied with the difference of tissue, the place and the time of taking the sample. The highest level of copper in Liver’s tissue was recorded in Dokan district during November, while the lowest level of copper in the meat tissue was recorded in Said Sadiq district during November. The triple interference for the study factors, also affected the level of Zinc in different sheep tissue were the amount varied by tissue difference, place and the time of sample taking. Highest level of Zinc was recorded in kidneys tissue, in Sulaimanyah city Centre during October, while less amount of Zinc was recorded in liver’s tissue in Said Sadiq during October. The triple interference within the study’s factor, significantly affected the amount of cadmium. The amounts were varied by difference of tissue, place and time of taking the samples. Highest level of cadmium was recorded in the meat tissue, at Sulaimanyah city Centre during October, while less amount of Cadmium was recorded in liver’s tissue, Said Sadiq district during October. The triple interference did not affect significantly the amount of Lead and Cobalt in different sheep’s tissue.
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Interesting research!
Did you make a conclusion which sousrces hade made an effect of meat toxication by heavy metals?
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I'm trying to work out which cell culture medium is the most appropriate to culture these 2 cell lines. I have never worked with non-cancerous cell lines, and I am asking myself if there are easier culture protocols that the ones stated in the ATCC web page.
  • NL20 should be cultured with HamF12, but then having to add up to 8 complements.
  • THLE-3 cultured with BEGM Bullet Kit, but I find it quite expensive, so searching I found some articles where they culture them with other similar mediums (# 511-500 , CellApplication) or even simpler and cheaper options as RPMI, DMEM, EMEM but they do not specify much...
Has anyone used different mediums than the ones in ATCC or has a protocol they are willing to share?
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Hello everyone,
I am working with Precision Cut liver slices of 250 µm. These need to be processed for FFPE. As you can imagenie they are very fragile and tend to break when I embedd them in Paraffin after fixation and dehydration.
Does someone have experience embedding thi tissues in HistoGel before Processing? My Plan is to fix the slices in Formalin over night, then embedding them in Histogel followed by processing for FFPE. Would it make sense to fix the HistoGel, too?
How does HistoGel behave when cutting?
Thanks in advance :)
Best, Franziska
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We routinely fix and paraffin embed 300um tissue slices with standard methods, and don't have issues with the tissue breaking. You don't mention when the breaking occurs, but if it's when you're doing your sections, it suggests that your tissue is too dehydrated, which can make it brittle. Long fixation in standard formalin (ie. 4% formaldehyde) does not make tissue brittle, it's the processing that does. I would check your processing schedule, and discuss with experienced histotechnologists whether it should be adjusted. Processing schedules made for standard tissue samples might be too long for these small tissues.
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Dear All,
Can microsomes be obtained from liver tissue without ultracentrifugation for in-vitro drug metabolism?
Thank you.
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Thank you, Malcolm and Daniel, for sharing the related connections.
Kind regards
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My lab has one or two vials that we obtained from a professor that left, but we'd like to get a more official stock. Are there any companies selling them?
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I have seen some dark areas in my sirius red staining of CCl4 treated mouse liver.
However, I am not sure what are these areas indicating.
It would be nice if some histology expert could help me identifying them.
Thanks a lot in advance.
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The “shadow region” is an extended area of fibrosis. To perform the polarization study you need to have a DIC microscope (differential interference contrast).
a typical procedure for manipulation of the polarizers starts on page 3 of the reference below.
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Many patients gets liver necrosis, and other liver toxicity suspected to have come from prescribed drugs mainly anti-drugs. There are many causes but specifically I wanted to localize on because it has risen many questions even from local population after being initiated to those drugs they developed liver ailments.
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procedures used to diagnose toxic hepatitis include:
Physical exam.
medical history.
Blood tests.
Your doctor may order blood tests that look for high levels of certain liver enzymes. ...
Imaging tests. ...
Liver biopsy.
toxicity occurs through mitochondrial damage resulting in lactic acidosis and hepatic steatosis. Protease inhibitors induce DILI in 6–11% of patients, but the incidence is significantly increased in HBV or HCV co-infections and alcohol consumption
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Mass (kg) X acceleration = Inertia which is the same as the intersection of the base. The product of inertia if we multiply it by the height we find the overturning moment of the column. If we have a wall that has a double lever arm (except for the lever arm of height and that of width) then the product of the tipping moment is divided by the width of the wall and this will be the tipping moment of the wall. If the wall is anchored at its base, a reaction will be created to the overturning torque of the lever, which multiplies (as we have seen) the overturning forces, since, as the height increases, its overturning force also multiplies. If the anchor is at the bottom of the wall, the critical failure area will also appear there and the anchor point is also the lever of the wall. Question If the anchoring of the wall is not at its lower ends, but is at its upper ends. That is, if we place this wall on a machine - press and apply pressure to it, it will remain a lever arm or its mechanical condition will change; 1) Will we have a multiplication of the tipping forces as it happens when the anchor is applied to its lower extremities? 2) Will a critical area of ​​failure of right forces N (compression and tension) be created as it happens when the anchorage is applied to its lower extremities? In short, we know that the walls drop high torques at the base since that is where the reaction of any substandard anchorage is. If the anchoring is done on the roof (ie if pre-tensioning is applied between the upper ends of the sides of the wall and the foundation ground) it will lower torques at the base and will create or not a critical failure area;
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Just for some context, I'm isolating non parenchymal cells with Pronase+Collagenase D, which is a well known protocol in my lab. I use DAPI as a viability dye in my FACS panel but I encounter some issues as it also stains high DNA content cells or mitotic cells, representing around 20% of the cells (see picture). No solution in the protocol contains detergent that could permeabilize the cells, and the mouse used is a classic healthy C57 so mitotic cells are not expected, at least to that extent. Did anyone encountered the same issue in the past? Thank you
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Reduce the concentration of the DAPI
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I fed this mouse normally for 6 days (control group). When I dissected this mouse, I found a white ball on the surface of its liver. What is it?
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Likely to be a hydatid cyst
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Hello!
How can the total amount of lipids in the liver be biochemically determined without the use of chloroform and methanol?
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The relationship between lipid profile and severity of liver damage in cirrhotic patients
This article might help you.
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I am currently amplifying a 2kb fragment from genomic DNA from mouse liver tissue. I am not sure if the DNA sample is of good quality or maybe it is even broken into fragments.I learned that I can check this with a gel elctrophoresis. How much DNA and loading dye should I add in the well?
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Elena,
Since your DNA amplicon size is 2 kb so, you can use 1% agarose gel electrophoresis.
Loading amount: Mix 1-2 microliter loading dye with 5-8 microliter DNA product and load into gel well
Ladder: Use 1kb plus ladder.
After completion of run, take gel image and compare your target band with ladder.
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Hello everyone!
I need to grow human liver cells (more specifically hepa RG cells) in a CO2-independent manner. Until now, I have used a Williams medium to grow them, but it has a bicarbonate buffering systems so it requires a controlled CO2 atmosphere. Does anyone have experience in growing these or other hepatocyte cell lines in CO2-independent media such as the Leibovitz L15 medium?
Thank you for your help!
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Hello researchers,
This is my first time working on monocytes, and I plan to deliver a drug to liver monocytes. Could you please suggest to me a way deliver this drug specifically to the liver monocytes?
I have considered exosomes and hydrogels, but I don't know how they specifically deliver to the monocytes! I am looking forward to your experience on this topic.
All the best.
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Dear Elham
You are welcome.
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I am working on qPCR of cDNA from liver in various states of health and disease, and our housekeeping genes show a fair amount of variation between disease states and other groupings. Which housekeeping genes do you use for liver research?
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I am currently doing a literature review, which is partly on fat-soluble vitamin metabolism in chickens. However, almost all recent research I can find on fat-soluble vitamin absorption & metabolism is focused on mammals. I was wondering how comparable fat-soluble vitamin absorption & metabolism is in e.g. humans vs. chickens. Is research done with Caco-2 cells or in mice, for example, translatable to chickens? Additionally, does this differ between parts of the body? Is there a big difference between how strongly e.g. enteric absorption and liver metabolism are conserved?
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For patients taking Glutathione supplementation for Ulcerative Colitis or other disease, will this at all negatively impact the liver's ability to produce it's own Glutathione?
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Excellent recommendations
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Hi all,
Now, I am doing quantification of infiltrated leukocytes in the liver by flow cytometry and qPCR.
Is perfusion needed before I harvest mouse liver?
Are intra-sinusoidal leukocytes "infiltrated-leukocytes"?
If so, I wonder liver perfusion washes out most of leukocytes in the liver sinusoids.
Thank you for your help.
Sincerely,
Park
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If you intend on specifically quantifying infiltrated leukocytes in the liver parenchyma, a perfusion is highly recommended prior to processing the tissue for analysis.
We perfuse (transcardially) at 4 mL/min (or 4-5 mL/min if perfusing through the portal vein) to wash out cells from the sinusoids (RBC or other blood cells). However if the leukocytes are activated and adhere to the endothelia there, they will not be removed by perfusion.
In case you already know how to discriminate between infiltrated vs adhered leukocytes transcriptionally, you can run both these primer sets on the tissue lysate to account for the activated vs activated and migrated populations of leukocytes.
But yes, in either case a perfusion is highly recommended (both for flow cytometery and qPCR analysis).
Hope this helps
Best
Aparajita
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Looking for a normal liver line to compare drug toxicity with HCC lines.
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Hey Francesca,
I am wondering wether you found an answer to your question or not, since I am looking for the same information.
Kind regards.
Anne
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Would I be able to do immunofluorescence on my liver tissue if I do not perfuse before harvesting? I don't intend to use the 488 channel.
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Use the scissors to cut the abdominal skin vertically to allow drainage of blood and perfusion liquid. Then squeeze the effluent blood vessel a few times with the straight forceps to inflate the liver, ensuring that all of the blood has drained out, and perfuse the liver with PBS until the liver tissue blanches
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I have a batch of fish liver samples than I would like to preserve for internal controls during EROD assays. Would lyophilizing the fish liver samples prolong the length of time that I can keep them stored -80C without affecting EROD activity?
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Juan C Pérez-Casanova - I would not recommend freeze drying the basic liver samples - that's a complex tissue to freeze dry and preserve the enzyme. Solid tissues, even small samples, generally take days to freeze dry, and then the challenge is to reduce the residual moister to <2%. Freeze drying can preserve enzymes well, when properly prepared, most PCR reagents and enzymes are freeze dryed, for example. I suggest that you extract your enzyme, aiquot in buffer and freeze dry the aliquots, then store in the freezer. Do watch out though, even freezing can affect activity as there can be a pH shift in buffer, when you freeze.
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Persistence of Hepatitis B surface antigen (HBsAg) in the serum is one of the defining features of chronic hepatitis B. Regardless of the exact phase and the eligibility for treatment, many patients with chronic hepatitis B are concerned whether their blood test will clear after treatment. Therefore, can it really clear?
If so, which specific treatment is documented to do that? Even after clearance of the viral marker, does the risk of liver damage decrease?
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Hepatitis B never really goes away; once you clear HBsAg, your risk of liver damage and liver cancer diminishes tremendously. It's worth a celebration, but you need to continue to be monitored as you age
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I recently did WB to check the pCREB/CREB after glucagon stimulation in mouse liver, but I got no band both for pCREB and CREB. I used RIPA buffer with proteinase and phosphorylase inhibitor cocktail to do the liver protein extraction. Mix the liver lysates wtih Lameli buffer and BME, and run with reducing SDS-PAGE. Does any one ever have the experience to do the WB for pCREB and CREB in mouse liver? Do I need to do the nuclear extraction?
I am not sure if the problem is the specifity of the antibodies or the sample preparation.
Both pCREB (9198) and CREB (9197) antibodies are from CST.
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Instead of checking only the mRNA level, I want to check the active protein level of MMP-1 in Liver tissue from mice. How can I do that?
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The activity of MMP-1 and MMP-13 in blood and liver tissue measure by using the sandwich enzyme immunoassay technique with commercially available quantitative ELISA test kits. Amino acids were determined by automated ion-exchange chromatography.
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Hello
I want to measure the amount of HDL in the liver, but the existing formulas show me negative values. Is this normal?
Is there a specific formula for measuring HDL in the liver?
I triglycerides and LDL cholesterol levels in've got
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Hi!
I want to know what is the best method to lysis rat liver tissue in western blot method. I am using RIPA buffer and I am not getting results. Do you think we can use Rıpa buffer for liver tissue? As a result of my research, I saw that the Rıpa buffer is used, so I use the Rıpa buffer. The recipe for my ripa buffer: 150mM Nacl, 50 mM Tris (Ph 8.0) 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS. I use 500 ul of ripa buffer and 5 ul of protease and phosphatase inhibitor cocktail per 10 mg of liver tissue. Then I sonicate at 45% amplitude 3 times at 10-second intervals, for a total of 30 seconds. Then I centrifuge at 14000 rpm +4 C for 20 min. Do you think there is something wrong with my method? Thank you from now.
Didem KORKMAZ
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Looking at your buffer composition, I don't see anything intrinsically problematic about it, although given the high metabolic activity of liver, it is possible that your protease inhibitor/phosphatase inhibitor is inadequate. Which protease and phosphatase inhibitor(s) are you using?
The other potential problem point is sonication. I have never used sonication to homogenize liver - although it was a LONG time ago, the method of choice for homogenizing liver was a glass homogenizer known as a Dounce. There were even motor driven ones for tougher tissues such as lymph node.
The combination of detergents - NP-40/deoxycholate/SDS should be adequate to solublize any membranes that could cause loss of your protein of interest.
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In our mouse bladder and liver sections we see a great deal of folding (creases in the tissue coming out of plane from the slide). May be noticed as early as upon transfer to water bath: tissues of liver section appear to wrinkle or crease almost like shrinking (43C DI water). Next day resting slide for 10 minutes at 65C does not seem to significantly decrease folding.
Sectioning method uses paraffin block chilled in freezer and regularly chilled with ice pack. Regardless of new or worn blade portion.
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Robert Spitz thank you. The use of 20% ethanol is a conventional practice and not suggestive. Take your section to 20% ethanol and allow some minutes to stretch out the sections. This is done before taking it to your water bath. Skipping this method, bear in mind that whenever you notice folds in your sections on the water bath, you may be tempted to increase water bath temperature or elongate the time which is not too good for your section quality as "cracks" across the epithelial and connective tissues could be observed after staining, thus histologically, your slides (under the microscope) may be poor (quality-wise).
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Surface guided radiotherapy is the state-of-the-art imaging module for patients who underwent for the radiotherapy. Some commercial available SGRT modules work on the principle of rigid registration algorithm and others work on the basis of DIR algorithm for surface matching. So, which algorithm is best to use in clinics for SBRT of liver cases?
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There are numerous reports in the literature about the good performance of both systems: DIR-based (such as Catalyst) and rigid ragistration-based (such as VisionRT).
Some reposrts for rigid-deformation based systems:
1. Deantonio, L., Masini, L., Loi, G., Gambaro, G., Bolchini, C., & Krengli, M. (2011). Detection of setup uncertainties with 3D surface registration system for conformal radiotherapy of breast cancer. Reports of Practical Oncology and Radiotherapy, 16(3), 77–81. https://doi.org/10.1016/j.rpor.2011.02.003
2. Apicella, G., Loi, G., Torrente, S., Crespi, S., Beldì, D., Brambilla, M., & Krengli, M. (2016). Three-dimensional surface imaging for detection of intra-fraction setup variations during radiotherapy of pelvic tumors. Radiologia Medica, 121(10), 805–810. https://doi.org/10.1007/s11547-016-0659-9
And some for DIR-based systems:
1. Pallotta, S., Kugele, M., Redapi, L., & Ceberg, S. (2020). Validation of a commercial deformable image registration for surface-guided radiotherapy using an ad hoc-developed deformable phantom. Medical Physics, 14527. https://doi.org/10.1002/mp.14527 (In this paper they found that for the Catalyst system there is an improvement when using DIR compared to rigid).
2. Stanley, D. N., Mcconnell, K. A., Kirby, N., Gutiérrez, A. N., Papanikolaou, N., & Rasmussen, K. (2017). Comparison of initial patient setup accuracy between surface imaging and three point localization: A retrospective analysis. Journal of Applied Clinical Medical Physics, 18(6), 58–61. https://doi.org/10.1002/acm2.12183
A comparison of both systems with good and comparable results:
Laaksomaa, M., Sarudis, S., Rossi, M., Lehtonen, T., Pehkonen, J., Remes, J., Luukkanen, H., Skyttä, T., & Kapanen, M. (2019). AlignRT ® and CatalystTM in whole-breast radiotherapy with DIBH: Is IGRT still needed? Journal of Applied Clinical Medical Physics, 20(3), 97–104. https://doi.org/10.1002/acm2.12553
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I am using an enzymatic kit for Lipid analysis from liver tissue and I have trouble in converting the 0.018 mM of TG to mg/g of liver.
Please help me with this. The details of the experiments are given below.
Weight of liver tissue used =106 mg.
Briefly, the tissue was extracted using chloroform:methanol and filtered to make upto 6 mL volume with the same solvent.
Then, 250 μL was taken from it and added 250 μL 1X triton-100.
the solution was allowed to dry under Nitrogen gas and after complete evaporation of the solvent the residue was dissolved in 0.5 mL of distilled water (considered as the final volume for calculation by me).
50 μL of samples were used for the enzymatic assay.
The molecular weight of TG= 885.4 g/mol (as given in the kit manual)
I tried to follow the equation mass (mg)=concentration (mM)* volume (mL)* molecular weight (g/mol), which give an answer 75.5 mg/g of liver.
But I don't know if this is the right method.
Please help me with a detailed calculation if possible.
If I had made any error in this calculation please point that out.
Thank you.
I really appreciate and respect your expertise in this calculation things.
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Thank you so much everyone. Really grateful for people like you who share their knowledge and help other researchers. Thanks again.
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Pleas.. what is liver important on coronavirus .this question is clear....ok
I ask and I need known physiology in liver on virus and killed him
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Dear Ihab Asaad Altameemi if you want to know how COVID-19 affects the liver, please have a look at the following useful links entitled:
1. What to Know About Liver Disease and COVID-19
(updated February 8, 2021)
2. COVID-19 and the liver: What do we know after six months of the pandemic?
3. Impact of COVID-19 on liver function: results from an internal medicine unit in Northern Italy
This article is freely available as public full text on rResearchGate.
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i have problem in preparation of liver tissue with bad cell organelles and how i can adjust osmolarity
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Since there are a lot of steps in preparing liver tissue for TEM, it is difficult to say where you might be going wrong without more information about the protocol you are using.
I would first check that you are using the proper fixative. I use 2.5% glutaraldehyde buffered with a cacodylate-sucrose solution (0.1 mol L-1 sodium cacodylate and 0.1 mol L-1 sucrose, pH 7.6) and then fix at 4°C.
For the processing, I stain with 1% OsO4 first and then with 0.5% uranyl acetate followed by ethanol dehydration and embedding. There are wash steps in between of course, I use 0.1N acetate buffer. This protocol works well for me but there are other very good ones out there too. If you are able to find this book, then I recommend it highly: https://www.springer.com/gp/book/9780306477492
- Melissa
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hi,everyone:
I try to analysis cholesterol concentration in serum and liver recently , I see protocol and it says that the serum lipid extract sovlent do not need to add triton, but liver do.
I am curiously about why there are different?
Also, l analysis triglycerides concentration in serum and liver, both do not need to add triton.
l can’t understand why triglycerides do not need to add triton?
sorry for my poor grammar, i hope you guy s can understand what i mean 😢
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Dear Ting,
For plasma TG and Chol , you dont need triton x-100
however for liver lipid extraction , you need a so called Bligh&Dyer extraction.
After the extraction TG and Chol are in a chloroform phase , the tritonx100 2% is needed to resolve the lipid in a water phase .
Lipid in water phase are used in the comercial kits
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I have frozen sections of 4%PFA drop-fixed, 30% sucrose dehydrated fetal livers from E14.5 mouse embryos. I sectioned them both at 10 and 5 microns. When I performed the staining on the 10 micron sections the DAPI was extremely dense and therefore I went thinner on the next round. However in both attempts I could not detect any c-Kit signal despite confirming the antibody does work on whole embryo sections. I am using c-Kit eBioscience cat. 14-1171-82
My questions are:
1. Is it just a matter of hit or miss? To my understanding there is not a robust amount present in the fetal liver at any point in time so maybe it is just a matter of staining enough slides to catch some c-kit positive cells?
2. Is there something extra needed to be done as far as the tissue prep? Most protocols in the lit are very vague. The fetal liver is full of blood cells and maybe I should consider perfusing, but then is it possible I also end up flushing out my cells of interest (HSC)?
Thanks in advance!
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Hi Lissenya!
I'm currently trying to do something very similar and struggling with the same issue. Have you found a solution to your question?
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Hello, I am currently using a Cas9 knock in system that will be expressed in the liver. To do so STOP codon fl/fl Cas9 KI mice were bred with Albumin-Cre mice to generate heterozgous pups, that were then crossed. For future planning, is it reasonable to expand a Cas9 KI+/+ Cre+/+ colony and then breed these animals to a Cas9 KI+/+ Cre-/- mice? Then all pups will be Cas9+/+ Cre-/+. The experiment require a large quantity of male mice.
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There are two popular mouse lines "specifically" expressing Cre in hepatocytes, i.e. Alb1-Cre and Albumin-Cre mice.
The location of Cre in Alb1-Cre mice is yet to be reported (I did not know where it is by last year).
B6.FVB(129)-Tg(Alb1-cre)1Dlr/J Stock No: 016832 | Alb1-cre
The location of Cre in Albumin-Cre mice is known. You can find mice with Cre+/+
B6.Cg-Speer6-ps1Tg(Alb-cre)21Mgn/J Stock No: 003574 | Albumin-Cre
Its genotyping Protocol 26917: Standard PCR Assay - Tg(Alb-cre)21Mgn-Alternate 1.
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We used enzymatic digestion method (collagenase 4 & trypsin ), but the cell number was less, around 2 X 105. Since flow cytometric analysis that we are doing need more events, there is no other way other than increasing the number of cells. Is there any better way to do the digestion? I found that hyaluronidase can improve the cell number if used for digestion. Can anyone suggest an alternative?
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Alexandr Chernov We don't have MACS tissue processor. Could you suggest any method alternative to it?
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There is inverse relationship between protein intake and risk of heart diseases because cholesterol start to increase as liver and cell process fats have low efficiently ( liver and other other cell required protein to its function)
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Hello!
I've stained some bright field Arginase-1 Hematoxylin slides, but I'm having a hard time finding any computed histology predicates for evaluating the intensity of the cytoplasm verses the nuclei; as everything seems to be done manually.
I've attached some 20x images of the slides. But I'm concerned about edge detection of the cell and haven't had much luck with segmentation in ImageJ. I lose a lot of edges in-between some of the cells.
  • Does anyone have any other software or ImageJ plugins you'd recommend?
  • Any specific computed histology guides or methods of hepatocyte specific antigens or something similar?
  • Any other guidance?
Thank you for your responses.
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You might find CellProfiler useful. Agreed, the cell borders are very faint. If you exclude nuclei and quantify staining within cells, might not need to segment individual cells. Good luck.
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I looking for answer to above question
All comments are respect
Yours sincerely
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Zaim Gashi Thank you for comment and contribution. Really thanks for your support and kindness
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Dear researchers,
I will look at ER stress proteins from rat liver tissue by Western blot method. I found the my protein concentration to be 86.386 µg / ul. How many ug of protein should I choose accordingly? How many ul of Laemmli Buffer Solution (2x) should I add for loading? Is the concentration of the liver lower compared to other tissues? Should I be careful about this when determining the protein amount?
Thank you.
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I don't know what volume can your wells take and what volume you have loaded, but both water+laemmli and empty wells are next to highly saturated wells. It looks like there might be small leak from saturated wells to the empty wells. That can happen when you try to load high volumes, or if you move the tank after loading the samples.
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Hello,
are there any murine cancer cell lines out there that once injected subcu will metastasize to the liver? I know there is LLC and some melanoma cell lines that will go to the lung. Appreciate any input!
thanks
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4T1 cells (mouse)
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I want to do tissue subcellular fractionation of mouse liver. According to the method, the speed to separate ER is over 200,000g for 120 min (Song et al., 2006). However, liver exosome isolation only needs 100,000 x g for 70 min (Ishiguro et al., 2019). Thus, I'm wondering how to separate exosome and ER?
Song, Y., Hao, Y., Sun, A., Li, T., Li, W., Guo, L., ... & Wei, H. (2006). Sample preparation project for the subcellular proteome of mouse liver. Proteomics, 6(19), 5269-5277.
Ishiguro, K., Yan, I. K., & Patel, T. (2019). Isolation of tissue extracellular vesicles from the Liver. JoVE (Journal of Visualized Experiments), (150), e58649.
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Is your target EVs only? I used collagenase and DNase together with mechanical disruption (cutting) and checked possible contamination by calnexin or RPL5. You can get a purer EV yield by applying a sucrose cushion.
For more information, you can check my published paper
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Hi,
I have been doing Reverse Transcriptase qPCR on some liver mRNA samples. I am targeting liver inflammation genes like Col1a1, alpha SMA, IL6, IL1B and etc... Even though mRNA concentrations are good and primers are working, some samples are not working (difference between two samples are higher than 0.5) for a few runs in a row. After I started using triplicates of repeat samples instead of duplicates, I am getting more successful results. For analysis, I am considering the closest two and taking the average of these two samples. Does it sound appropriate in terms of data analysis ? Has anyone been doing this ?
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If you do triplicate runs, use the average of the triplicates. Making a general policy of discarding replicate data is a bad idea.
In practice, this comes with some (user-defined) case-by-case cut-offs.
If I got 22.2, 22.3 and 26.8 for triplicates, I would look at all the other samples, and if they were all hovering around the 21-23 ballpark, I would absolutely drop the 26.8 as a "John did some crap pipetting" error. If I got 22.2, 22.3 and 23.1, I would be more inclined to use all three, or repeat the run.
The issue really comes down to how much you can trust your data: with variable duplicates, either result could be the valid one. With variable triplicates, at least you usually have two that agree and one that doesn't. And if that isn't the case (the dreaded 22.1, 23.2, 24.5 result), then you re-run those samples (alongside trustworthy data so you can back-calibrate).
I would always recommend using triplicate wells, reserving duplicate wells only for genes that you have run countless times and found to be incredibly consistent. When you consider the economics, duplicate wells you have to repeat end up costing more than triplicates you don't.
I know others may argue in favour of a more rigid approach, but it's molecular biology: an innately messy process. Be informed by the actual scenario, rather than hard rules. A gene that gives 22.3, 22.5, 22.1 in untreated samples and 26.7, 25.4, 26.9 in treated samples is a gene that is clearly dropping in expression, whether you decide to trust that 25.4 or not, and (equally) the extent to which it is dropping is pretty similar either way. The biological readout is clear.
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Hi, im newbee for analysing gen expression data set and hope someone have som advise.
Im working on mice liver gen expression (mRNA) data sets generated with EdgeR - now I want to do some gen enrichment analysis - but is confused as to which methods are most robust and reliable (and to some extent user-friendly).
I have tried EnrichR, ROntoTools and GSEA - out of these, I think GSEA is the most difficult to understand.
So - what do you use? Do you have any tips for me?
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Hei! Stumbled across this :
will give it a try myself but the idea seems tempting in case one wants to give a short macroscopic snapshot (especially when comparing several time points and cellular fractions).
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I am working on liver disease classification using Machine learning. I would like to know is there any public dataset available for liver disease patients data which includes both liver functional test results and liver image of same patient.
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Dear colleagues,
I would like to ask you if anyone has experience with the fixation of whole rat organs (heart, lungs, liver, kidneys, brain, testes) in 10% buffered formalin.
Thank you,
Roman
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Should be fine in 10% formalin if not left for more than 48hrs. Then paraffin embed to store
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Which integrin subunit plays more important role in the liver? In connection with TGGB Community Verified icon Community Verified icon
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Dear Sir
Try to have a look at this article. It's all about ILK.
hope this will help you.
Regards
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