Hi, it is my first time to do cryosections and immunostaining for mouse pancrea, and face a lot of technical issues. I will use GFP to stain the neural cells in pancreas, and have no idea how to do the immunostaining. Also, if I want to freeze the pancrea and do cryosections, how thick should I slice the tissue? Some protocols suggest 5 μm, is it too thin? Thank you for every suggestion!!
5 um sections are fine for immunostaining, You can also use paraffin embedded sections for both immunostaining and immunoflouresence, but you have to deparaffinise and hydrate the sections before staining process in case for paraffin embed sections where as you can skip these steps in case of cryosections.Procedure can be fallowed from any published article.
1.Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue …SR Shi, ME Key, KL Kalra - Journal of Histochemistry & …, 1991
5 to 7um are good. If thinner is difficult to handle, go for 7um. if you can handle 5um, it is great.
I Freeze the tissue in Optimal Cutting Temperature (OCT) embedding media with dry ice and then do cryosection. I am wondering how do you usually do the immunostaining? Subhash C. Juneja
Uday Kumar Chekkilla Hi, I use OCT to embed the pancreas instead, but can not find the protocol. Have you ever done this? And do you think whether the staining steps are similar with paraffin embedded method? Thanks in advance!
That is OK, you are putting tissue in OCT and immediately throw the mold on Dry ice and orient the tissue immediately than place in the freezer at -70 C at least or in Liquid nitrogen or thwing directly in Liquid nitrogen, that is OK too (in this case, orientation is not possible). Placing of dry ice is helpful first so that you can orient the tissue while you can see.
Second method is that
*you fix the tissue in 4%PFA (freshly made) or freshly diluted PFA (Source: EM sciences, you can get 16% in glass capsules). Fix tissue for one hour at 4C.
*Wash tissue two times in PBS to get rid of Fixative
*Place the tissue in 15% sucrose, till tissue settles down at the bottom of the tube (you may use cold room) with very slow shaking
*Transfer tissue in 30% sucrose, till tissue settles down at the bottom (you can use cold room and slow shaking)
This sucrose step may take upto 24hr
*Pass the tissue thru 2/3 changes of OCT to get rid of sucrose solution on the surface of tissue
* finally place fresh OCT in cassette and add tissue in cassette and orient it while putting the block on dry ice.
After that steps are same as I said in the first method
Advantage of Cryosections is that you do not need antigen retrieval over paraffin-embedded tissues
A ppt is enclosed for your knowledge and for your more thinking