[Histonet] Re: Masson's trichrom - problem with nuclei staining

[Andrea Marion]

Hi Itai,

I am interested to hear if you've resolved this problem.  We use the same
kit to stain mouse heart and embryo sections, PFA fixed. Our protocol is
similar to the one you mentioned. I cannot get nuclei staining with this
method either. The nuclei are well stained (ie black) up to the PMA/PTA
step, but during that step the nuclear stain is completely removed. I
cannot shorten the PMA/PTA step without negatively effecting the collagen
stain.

I have in our original protocol that the Weigert's working solution is
good for one month, but I cannot recall if this is from Sigma's
specification sheet or a personal observation from a lab member.  However,
from reading online some say it is good up to 4 months, others say it
needs to be prepared fresh each time. I have tried fresh preparations with
the same results.

My instinct is that something is off - the staining is just not stable
enough to withstand the subsequent acid steps in Masson's trichrome. Can
an expert weigh in on this? Is there a way to strengthen nuclear staining
from Weigert's?

Sigma's formulas and usage recommendations are:

Part A: 1% w/v certified Hematoxylin in ethanol
Part B: 1.2% w/v Ferric chloride, 1% v/v Hydrochloric Acid

Combine equal volumes Part A and Part B, stain sections for 5 minutes.


Andrea Marion
Graduate Student
University of Illinois at Chicago
amario3 /at/ uic /dot/ edu


Itai Moshe itai.moshe <@t> mail.huji.ac.il
Wed Sep 15 11:34:51 CDT 2010

Hi Histonet's

I'm using Masson's trichrome to stain paraffin mice diaphragm fixed with
PFA.
I'm using this protocol:
http://www.bcm.edu/mcb/rosenlab/index.cfm?pmid=12997
<http://www.bcm.edu/mcb/rosenlab/index.cfm?pmid=12997>With sigma masson's
kit (#HT15) and Weigert's Iron Hematoxylin Set (#HT1079)
The staining is beautiful, but i can't see the nuclei good enough.
1) Is there a way to enhance the nuclei staining ? (the nuclei is the only
reason that i"m not using the simpler sirius red and fast green staining.)
2) What is the meaning for washing in running tap water washing, is it done
by putting the slides in a  jar with simple tap water for a few minutes ?

Thank's
Itai

P.S
By mistake I've post this before in another message with a wrong title.
please respond to that message, so the title will be o.k for future archive
searching.