Hi

You can test your staining solutions/kit  in other sample section. I think your staining kit is expired.

good luck

Hi Clémence,

Please indicate how old is the embryo, and why you are using cryosections. I have used this staining on paraffin sections of the ostrich embryo and the results were good.

good luck

Thank you for your answers

I don't use a kit but home-made fresh solutions.

Actually I need to stain teratoma cryosections (fixation 24h in 4% PFA) that may contain any kind of tissue. The aim is to check if there is ecto/meso/endoderm-like differentiation. The first try gave weak and non-reproducible staining, so I decided to test on tissue cryosections that contain all types of tissue for sure (muscle, digestive tract, skin...). I happen to work on chicken embryo so I took what I had in the lab, incubated eggs with 15 day-embryos. We don't use paraffin embedding here so I embedded in OCT which I know well.

I'm going to test the post-fixation in Bouin then (picric acid is supposedly forbidden here but we have some).

I stained the trichrome before in paraffin section fixed with Bouin and it was very good.

you can try to fix some embryos for paraffin

Hi Clémence,

I think the fixation time for 15-d-old chick should be at least 48 h at room temp in 4% PFA. Note that when your fixative is not Bouin's solution, post-fixation of sections in Bouin's solution is necessary (few drops of Bouin's s. on each slide). See this web page: http://stainsfile.info/StainsFile/stain/conektv/masson.htm

good luck

Dear Clemence,

I have also only used Masson's trichrome stain (homemade, not a kit) on paraffin sections.  Since my tissues were fixed with formaldehyde-based fix, I had to use postfixation with Bouin's solution, as Morea suggested.  

However, there is a thread here on RG that suggests post-fixing the sections in Acetone for 10 min at -20 C after fix in PFA.  I've never tried this on frozen sections, but it might be worth a shot.  At least it's less nasty than the hot Bouins!

Good Luck,

Jill

https://www.researchgate.net/post/Has_anyone_performed_trichrome_or_picrosirius_staining_on_cardiac_frozen_sections

Dear All,

Thank you for all the answers. I had read a lot on the Stainfile site actually before starting and tried to treat with Bouin without noticing enhancement of stainng, but this was not on the best sections, I'll try on the embryo section in which I'm sure that there are all tissues.

I know that post-fixation and mordanting are quite different. What I thought was that fixing (or pos-fixing) with a different fixative may change the "reactivity" of the tissue, even it is not chemically true "mordanting". I mean, some fixative are known to solubilize fat or make some molecules more accessible. With immuno-detection some epitopes need to be fixed with PFA and others with methanol-acetic acid for example, because methanol coagulates proteins that mask the epitope I guess. Bouin is indeed not only a fiwative but also a mordant, acetone is fixative but it may have an effect too, it's worth trying.

CK

I had the opportunity to fix chick embryos directly in Bouin for 24h to do cryosections The staining is better but still I don't see much contrast between the purple and the red, most structures that are not green are purple-reddish. Tissues are shrinked and the future bones tend to get away from the section. I also treated my PFA-fixed sections with Bouin as Morea suggested and indeed the staining was much better than on non-treated tissue, it's a good method to "rescue" tissue that has not been fixed with Bouin. However, here also the muscles were not red, maybe they are not in embryonic/fetal tissue?!? And also, I found different recipes for the Bouin, some using formaldehyde, some other formalin without indicating the final concentration of formadehyde, which varies according to my calculations from 2 to 10%. Probably not important if tissues are fixed several days but yet I'd like to be more precise to write down the exact composition of the fixative.