Hello, I have been working with H&E staining of mouse muscle (lower limb muscles - gastrocnemius, soleus, tibialis anterior) but seen to have issues with sample preparation. Well, I am guessing that issue is fixation/sucrose based, but I hope to gain a more expert opinion here.
My samples seem to have large spacing between cells and fibers. The spacing size seems too much to be physiological. I have tried dissection -> fixation (PFA 4%) -> sucrose (30%) -> freeze in OCT (the results show in pictures 1 and 2). I have also tried dissection -> freezing -> fixing (4% PFA), shown in picture 3.
Any recommendations of altered protocols to get my slides closer to physiological muscle cells/fibers?
Sucrose will decrease water content within the tissue, which might lead to some the shrinkage you are seeing.
I suspect the cause is sucrose… The other steps of your procedure are identical to what I have worked with, barring the sucrose processing (and I’ve never had any issues like this.) I think you could probably try either eliminating the sucrose step, or alternatively try reducing concentration and/ time for the same. All the Best!
