[Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC! (Maria Mejia)
Maria, This may sound simplistic but I find that if I have problems such as this the first thing I do is try a new batch of the staining reagents. If you haven't had problems before then something must have changed either in your protocols or your reagents. It could be that your heat retrieval reagents are too old or contain detergents that remove some of the proteins you are looking for. Some of the proprietary heat retrieval reagents that allow you to heat retrieve without having to dewax by going through xylene have been shown to change the nuclear staining pattern and create what appear to be nuclei that are full of vacuoles. Also if the celloidion is not completely removed during your staining it may be stopping any of the higher molecular weight stains from penetrating the cells. Try leaving your sections in acetone for a number of changes to ensure full removal of the celloidion. Regards Steve Weston University of Tasmania Breathe-Well CRE Lab Manager 0408990859 University of Tasmania Electronic Communications Policy (December, 2014). This email is confidential, and is for the intended recipient only. Access, disclosure, copying, distribution, or reliance on any of it by anyone outside the intended recipient organisation is prohibited and may be a criminal offence. Please delete if obtained in error and email confirmation to the sender. The views expressed in this email are not necessarily the views of the University of Tasmania, unless clearly intended otherwise. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!
Maria, I. Think you may have a pH issue. High pH results in reduction of protons, H+, effects dye structure and can cause light to no staining after bluing. If you are using hematoxylin w/ aluminum, most popular, decreased pH = decreased intensity. Acid breaks the Al+3. Check your ph throughout the process. Sounds like something has changed. Good luck. Sent from my Windows Phone From: Morken, Timothy via Histonet<mailto:histonet@lists.utsouthwestern.edu> Sent: 1/4/2016 9:20 AM To: Maria Mejia<mailto:mbmph...@gmail.com> Cc: Histonet<mailto:histonet@lists.utsouthwestern.edu> Subject: Re: [Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC! Maria, If the counterstain is good when done before IHC stain and poor after it sounds like proteins are being extracted during the IHC processing and staining. Have you tried staining sections after each step of the IHC process to isolate the point the stain becomes weak? Tim Morken Pathology Site Manager, Parnassus Supervisor, Electron Microscopy/Neuromuscular Special Studies Department of Pathology UC San Francisco Medical Center -Original Message- From: Maria Mejia via Histonet [mailto:histonet@lists.utsouthwestern.edu] Sent: Sunday, January 03, 2016 8:29 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC! Happy New Year Everyone, I'm the lead histologist working in an IHC based research lab focused on early stages of Alzheimer's disease. We work on paraffin sections processed & cut from 600um celloidin sections. Including a lot of 60um cellodin sections from whole human brainstem. For years, everything has going good regarding counterstaining after single & double IHC staining on 60um free-floating sections. However for the past two months we've struggled to achieve good visible counterstaining on IHC sections to count the stained neurons - to see clearly the nucleus & nucleolus! For a number of years, Gallocyanine was our choice of counterstain after IHC. Now, it's NOT working (neurons not stained visible enough to count). We've also tried cresyl violet counterstain - staining too weak! In both counterstains, we modified the staining protocols quite a number of times to get good visible staining - nothing!!! Strangle because we get lovely counterstained neurons with NO IHC staining on our 60um sections, but as soon as we take the sections through the IHC protocol e.g. antigen retrieval, antibodies & chromogens - counterstain is too weak! Our paraffin IHC sections work & look wonderful! Now, my PI wants to try methyl green counterstain, however I think we'll have the same problem. Here's what I need help with: 1) Can someone please explain the reason or theory behind the failure of counterstain uptake by cells such as human neurons on 60um celloidin sections? 2) Can anyone please offer staining protocols that use alternative dehydration & clearing reagents. I've been using alcohols dehydration (96% & 100%) without success as well as clearing with xylene - which hardens the tissue if left too long in this reagent. I was thinking of perhaps using acetone instead of alcohols & maybe using a methyl salicylate or chloroform. Thoughts anyone? I wish Dr Chris van der Loos was still with us. I'd dearly like to hear from anyone who can help with this issue - Gayle Callis, Terry Johnson, Dr Hohn Kiernan et al. Any assistance anyone can provide will be greatly appreciated! Best Maria Mejia UCSF Memory & Aging Department San Francisco, CA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!
Maria, If the counterstain is good when done before IHC stain and poor after it sounds like proteins are being extracted during the IHC processing and staining. Have you tried staining sections after each step of the IHC process to isolate the point the stain becomes weak? Tim Morken Pathology Site Manager, Parnassus Supervisor, Electron Microscopy/Neuromuscular Special Studies Department of Pathology UC San Francisco Medical Center -Original Message- From: Maria Mejia via Histonet [mailto:histonet@lists.utsouthwestern.edu] Sent: Sunday, January 03, 2016 8:29 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC! Happy New Year Everyone, I'm the lead histologist working in an IHC based research lab focused on early stages of Alzheimer's disease. We work on paraffin sections processed & cut from 600um celloidin sections. Including a lot of 60um cellodin sections from whole human brainstem. For years, everything has going good regarding counterstaining after single & double IHC staining on 60um free-floating sections. However for the past two months we've struggled to achieve good visible counterstaining on IHC sections to count the stained neurons - to see clearly the nucleus & nucleolus! For a number of years, Gallocyanine was our choice of counterstain after IHC. Now, it's NOT working (neurons not stained visible enough to count). We've also tried cresyl violet counterstain - staining too weak! In both counterstains, we modified the staining protocols quite a number of times to get good visible staining - nothing!!! Strangle because we get lovely counterstained neurons with NO IHC staining on our 60um sections, but as soon as we take the sections through the IHC protocol e.g. antigen retrieval, antibodies & chromogens - counterstain is too weak! Our paraffin IHC sections work & look wonderful! Now, my PI wants to try methyl green counterstain, however I think we'll have the same problem. Here's what I need help with: 1) Can someone please explain the reason or theory behind the failure of counterstain uptake by cells such as human neurons on 60um celloidin sections? 2) Can anyone please offer staining protocols that use alternative dehydration & clearing reagents. I've been using alcohols dehydration (96% & 100%) without success as well as clearing with xylene - which hardens the tissue if left too long in this reagent. I was thinking of perhaps using acetone instead of alcohols & maybe using a methyl salicylate or chloroform. Thoughts anyone? I wish Dr Chris van der Loos was still with us. I'd dearly like to hear from anyone who can help with this issue - Gayle Callis, Terry Johnson, Dr Hohn Kiernan et al. Any assistance anyone can provide will be greatly appreciated! Best Maria Mejia UCSF Memory & Aging Department San Francisco, CA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!
Happy New Year Everyone, I'm the lead histologist working in an IHC based research lab focused on early stages of Alzheimer's disease. We work on paraffin sections processed & cut from 600um celloidin sections. Including a lot of 60um cellodin sections from whole human brainstem. For years, everything has going good regarding counterstaining after single & double IHC staining on 60um free-floating sections. However for the past two months we've struggled to achieve good visible counterstaining on IHC sections to count the stained neurons - to see clearly the nucleus & nucleolus! For a number of years, Gallocyanine was our choice of counterstain after IHC. Now, it's NOT working (neurons not stained visible enough to count). We've also tried cresyl violet counterstain - staining too weak! In both counterstains, we modified the staining protocols quite a number of times to get good visible staining - nothing!!! Strangle because we get lovely counterstained neurons with NO IHC staining on our 60um sections, but as soon as we take the sections through the IHC protocol e.g. antigen retrieval, antibodies & chromogens - counterstain is too weak! Our paraffin IHC sections work & look wonderful! Now, my PI wants to try methyl green counterstain, however I think we'll have the same problem. Here's what I need help with: 1) Can someone please explain the reason or theory behind the failure of counterstain uptake by cells such as human neurons on 60um celloidin sections? 2) Can anyone please offer staining protocols that use alternative dehydration & clearing reagents. I've been using alcohols dehydration (96% & 100%) without success as well as clearing with xylene - which hardens the tissue if left too long in this reagent. I was thinking of perhaps using acetone instead of alcohols & maybe using a methyl salicylate or chloroform. Thoughts anyone? I wish Dr Chris van der Loos was still with us. I'd dearly like to hear from anyone who can help with this issue - Gayle Callis, Terry Johnson, Dr Hohn Kiernan et al. Any assistance anyone can provide will be greatly appreciated! Best Maria Mejia UCSF Memory & Aging Department San Francisco, CA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet