I am interested in a good protocol to dissect and prepare mouse intestine for immunohistochemistry. I have all the possibilities from perfusion of the animals etc.
What protocol of fixing/freezing the tissue can I use?
Thanks for your help
I think it greatly depends on which antibody you are planning on using. We have previously performed e.g. CD45-staining in formalin-fixed, paraffin-embedded sections. If you are unsure of which stainings you are planning on doing, I recommend to prepare for all kinds of staining. For each mouse, immediatly after sacrification, take small pieces (e.g. 3-5mm long) of intestine and freeze in liquid nitrogen, then embed in OCT-compound and store at -80. Then take another few pieces of intestine and put it in formaline, dehydrate and embed in paraffin. I would also recommend to take another piece and freeze again in nitrogen and store native in tube at -80°, so you also have material if you'd like to do an ELISA or WB from the tissue (you never know...). That way you are prepared for all potential stainings or analyses you might want to do. Antigen-retrieval and staining protocol etc. of course depends on the used antibody.
As Vera said, the method of preparation depends on what you intend to stain the tissue for. Formalin-fixed, paraffin-embedded (FFPE) tissue is always easier to use and section and provides much better tissue morphology than frozen sections, but some IHC protocols do not work well with FFPE tissue, so it depends on what IHC you intend to do. Preparation of the intestine will also depend on which region you are interested in, and what you want to evaluate. Taking some short 1-2 cm segments of intestine can work for basic evaluation, but you should be aware that morphology of the small and large intestine will vary according to what level you are looking at. Another approach is to 'swiss-roll' the entire intestine (small and large done separately) and embed that flat in one cassette, so that you can see the entire length of the intestine in one section. That works well, but may not be ideal if you are doing more pointed things, like measuring villus lengths. So really it depends on what you are evaluating.
I am interested to stain and investgate some proteins within the smooth muscle of the intestine. We use those antibodies mainly in frozen PFA fixed brain or spinal cord slices and I never tried them in FFPE tissue. I will try with PFA perfused tissue soon and check if it works in my hand and with my regular staining protocols.
Thanks a lot for your advice