Our current protocol has us fixing slides (cryo or paraffin) for 24 hours. It was just brought to my attention that this is wrong, but I was not told how long is correct. I would love to know what others do for their protocol.
How long are you fixing the slides in Bouins Mordant before doing a masson's trichrome stain?
Thank you!
I think 24 hours in Bouins are correct, but you can also use formalin and start the staining with 2 hours in Bouins at 60°C. This should bring a similar effect.
If you fix in Bouin you don't have to mordant the slides again. You can start with the iron hematoxylin.
BTW mordant is the wrong description of the effect. It is rather a retrieval of binding sites for the dyes. This is necessary because formaldehyde binds to the same sites and "blocks" them.
Hi =)
From my experience we only use Bouins to fix bone marrow and we fix the tissue sample before embedding and cutting then for 24hour.
I've done quite a few Masson's Trichrome stainings (mainly in paraffin but also in cryo) and I don't have any special fixation protocol, just the 24 hours in formalin before embedding that's the usual protocol for every sample.
Hope I could help.
Good luck =)
I think 24 hours in Bouins are correct, but you can also use formalin and start the staining with 2 hours in Bouins at 60°C. This should bring a similar effect.
If you fix in Bouin you don't have to mordant the slides again. You can start with the iron hematoxylin.
BTW mordant is the wrong description of the effect. It is rather a retrieval of binding sites for the dyes. This is necessary because formaldehyde binds to the same sites and "blocks" them.
Gudrun,
Thanks for your answer. This is what my protocol states as well. I appreciate your answer!
The Bouin fixation is very good. The time of fixation will depends up on the thichness of the tissue and the age of embryos if you are using different ages.
I think form 18- 24 hrs will be enough for the fixation
We also used to fix overnight in Bouin but, as Manal stated, it is dependent on the thickness of the sample. Are you asking because you are having some specific difficulties? Sometimes people will claim that you should or should not do things based on no scientific evidence but just based on the "that's the way it's always done" principle. Not quite a fundamental law of physics!
Nicolas - no, I've had no issues however, my supervisor was displeased when he found out that I was overnight fixing. My samples are typically 10 um for cryo and about 6 um for paraffin. He seemed to think that this was too much or that there was no reason to fix in Bouins at all. So I wanted to find out how other people do their stains. It seems the overnight is pretty standard.
I really appreciate everyone's input. It's very helpful to hear how others do this.
I actually read a little too fast. Did not realize you were fixing sections and not tissue. We used Bouin for fixing tissues but not afterward on sections for the Masson's staining. On sections, overnight is probably overkill since the penetration is not an issue. I would guess one hour is enough. There is a lot of people that are concerned with overfixation when immunostaining is concerned but for Masson's, I would not thing that there is much risk of that. It might just be more efficient to fix only one hour.
Well, it looks like I have some experimenting to do. Thank you everyone, your advice is invaluable.
Hi Tanya,
I fixted the tissue (heart, testis) even 48 hours. Masson staining was great. This fixative is safe for tissue. I use it to cryo (-25 or more) and paraffin embedding.
Best Regards
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