[Histonet] Problem with Masson's Trichrome

[Bdeer24 <@t> aol.com]

Hi Everyone!

I was recommended to try the Masson's Trichrome stainings on the slides of uterine tissue that I have been collecting (Previously I had only performed Hematoxylin & Eosin stains). This is a new method to our lab and thus far, staining has not gone well. Although my sections contain a significant amount of muscle, I have only been able to see the aniline blue stain. On a couple of the slides, there was a slight dark purple coloration on the epithelial lining. 

I have noticed upon comparing the protocol we received to others found on the internet that ours lacks the step in which  the slides are placed in Bouin's fluid. There was also no specification in our protocol for the type of hematoxylin, so we had been using the same hematoxylin (Harris's modified with acetic acid) that we use in our H & E stainings. I was wondering if any of these might have led to the stainings not going as planned. The tissue sections also looked slightly brittle, perhaps due to a harsher stain. I am wondering if this has anything to do with my fixation process in which I use a 4% paraformaldehyde/PBS mixture, ethanol and xylene steps, as well as embedding the tissues in paraffin. 

Is Bouin's a crucial step, would I have better staining with a different tissue fixation, or should I be looking in a different direction? Any suggestions would be very much appreciated!
Thanks!

Amber Greenbank

Arizona State University