We have recently collected perfused whole mount sections of the rat gut, containing the duodenum, ilium and colon. Following perfusion the sections were cut longitudinally but kept all in one piece. They were stored in 4% PFA overnight before being transferred to PBS with 0.03% sodium azide for a few days. We then transferred the sections to PBS with 30% sucrose for 48 hours. Following this the sections were frozen in one piece using OCT.
Can anyone suggest a good way to section this tissue and if there are any antigen retrieval steps which should be performed when doing immunohistochemistry? We are hoping to look for alpha-synuclein.
Thanks! Teresa
I've mainly worked with marine organisms (e.g. fishes, hagfishes, sharks), but I think the process is similar. In my opinion, it really depend on how robust your alpha-synuclein antibodies are. I typically embedded gut and other fixed tissues within paraffin and sectioned with a microtome at room temperature. Instead of antigen retrieval, I found background quenching to be most useful. I use sodium borohydride (1.0 - 1.5 mg/ml) mixed within ice-cold PBS for 10 minutes, 6 times, to do the trick. Hope this helps.
Hi Teresa,
There was no need to keep tissue in PBS for few days, 30-60 minutes is OK. Rest all steps are good. You have already oriented the tissue in OCT cryo blocks. You can cut. I always cut 7um thick sections in cryostat. If 7 is difficult to handle, you can cut 10um thick. Once you cut section, place slides with section/s in slide box and keep box on dry ice (or at -70C) till all sections are cut in one session. And store slide box at -70C. You can check each section under light microscope immediately taken out of cryostat if it has right orientation right away.
In cryo sections, antigen retrieval is not needed, you can proceed with immunohistochemistry or immunoflurescence what ever you had planned.
Once you start immuno, take the frozen section out at room temp, leave horizontal on slide stand, leave there for 30 minutes (even 45 minutes) so that section settles down and adheres to the slide after OCT melts down (otherwise your section will fall off the slide),
DO you know immuno steps or you are new to immuno? are you doing immunoflorescence or using DAB or other chromagen method. Than I can help you accordingly
Thank you both for your helpful responses! Subhash C. Juneja I usually work with brain tissue for immuno, I have never worked with gut tissue. We are some time away from starting the immuno but if I experience any issues once I get started I will reply to this question and if you are able to help out I would really appreciate it.
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