I'm recently working on the Gomori's trichrome staining on adult mouse limb. The whole lower limbs (skinned) were fixed in 4% PFA at 4C , decalcified in EDTA, embeded in OCT and cut as 20um sections. The morphology was pretty good in freshly cut sections. However, when I fixed them in Bouin's solution at RT overnight, the skeletal muscle in the sections shrank severely while the bones and connective tissue were fine. So anybody has any idea what went wrong in the Bouin's fixation?
The problem probably isn't the Bouin's fixative specifically. Most fixatives cause shrinkage of soft tissues, which can be very substantial, and can differ across different tuypes of tissues. I would guess the high collagen content of connective tissues, including bone probably shrinks less than the skeletal muscle tissue in your samples. Fixing at room temp overnight is a pretty hard fixation and I can imagine that it could have big effects on your tissue.
Does your staining require Bouin's fixative? Could you just work with the tissue sections without doing the additional Bouin's fixation step (or use a different type of histological stain to see the various tissues in your sample that doesn't require Bouin's fixation)? Is the Gomori stain absolutely essential for your work? Any of a number of stains will work on PFA-fixed tissue. Finally, If you absolutely need to stick with the Gomori stain and Bouin's fixation, could you shorten your fixation time to possibly minimize the differential tissue shrinkage?
Good luck.
Dave Sherry
Did you use isopentane freezing method while freezing the muscle? It usually preserves structure otherwise ice crystals cause problems
I agreed with the comment of Dr. Sherry. Just try to shorten the time of Bouin's fixation. How about only 30 min fixation, because tissues were already sectioned. T.K.
Dear Yue Lu
A procedure for a modified Gomorri's trichrome that doesn't use a Bouin's step was posted here on RG. Maybe that modification will work for you.
https://www.researchgate.net/post/Any_advice_on_Massons_Trichrome_stain_on_frozen_muscle_sections
Good Luck!
Jill
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