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

Sat Sep 18 13:19:54 CDT 2010

I do tap watewr because it works without wasting water and all of the
water running could bring out some chemicals that affect the subsequent
staining.  Truth is most peopl;e never consider nulcei as an impolrtant
step of the masson.  If you want to do a faster stain do the van Gieson.

Nick Madary, HT/HTL(ASCP)QIHC

Medimmune Histology Mgr, 

OMW, Area 4, Lab 2438

301.398.4745(vm)

301.398.6360(lab)

301.398.9745(fax)

________________________________

From: Itai Moshe [mailto:itai.moshe <@t> mail.huji.ac.il] 
Sent: Saturday, September 18, 2010 10:03 AM
To: deannal78 <@t> verizon.net; amario3 <@t> uic.edu;
histonet <@t> lists.utsouthwestern.edu; Madary, Joseph; plott <@t> uab.edu;
atliffjack <@t> hotmail.com
Subject: Re: [Histonet] Re: Masson's trichrom - problem with nuclei
staining

Hi, and thank's for the quick replay.

I'm using a fresh Weigert's prepared from the sigma kit every time.

maybe it is possible to modify and to use sirius red to stain for
collage. or to use methyl green for the nuclei.

Maye the bluing part is not done o.k, maybe like Mr' Madary offered, a
warm tap water will do the job ?

is it that important to use running tap water, and not just to immerse
the slides in tap water, because i don't have a proper bath to do this ?

Itai

2010/9/17 Deanna Rhoads <deannal78 <@t> verizon.net>

I do Weigert's staining for 10 minutes and use it for a week at the
most.  I, too, have heard conflicting times about the stability of the
Weigert's, but have the best results with using it no longer than a
week.

Deanna Rhoads HT (ASCP)

________________________________

From: Andrea Marion <amario3 <@t> uic.edu>

To: histonet <@t> lists.utsouthwestern.edu

Cc: itai.moshe <@t> mail.huji.ac.il
Sent: Fri, September 17, 2010 10:31:16 AM
Subject: [Histonet] Re: Masson's trichrom - problem with nuclei staining

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

010/9/15 Madary, Joseph <MadaryJ <@t> medimmune.com>

I never use the Masson and expect decent nuclear staining.  Do not
forget to mordant in Bouins, wash it out well, and the running water or
sitting in several changes of warm tap water is a bluing step.  

Nick Madary, HT/HTL(ASCP)QIHC

Histology Mgr, Medimmune

301.398.6360(lab), 4745(vm),9745(fax)

010/9/15 Patricia F Lott <plott <@t> uab.edu>

Make sure when you mix solution a and solution b, you have a very dark
purple color solution that smells fruity, like wine.  If not, check that
your solutions are not expired.  I buy the Weigert's kit (32 oz. of Sol.
A and 32 oz. of Sol.B) from Poly Scientific, and it works very well. 

The purpose of the tap water is for blueing, it will make the nuclei
very dark and crisp if you leave the whole slide rack in a sink with
running tap water for 10 minutes.

Best Regards,

Patty Lott

	2010/9/16 Jack Ratliff <ratliffjack <@t> hotmail.com>

	The tap water rinse is in reference to running tap water (not
directly on the slide sections) while the slides sit in a make-shift
water bath. This is done over a period of time to differentiate the
nuclei staining similar to a "bluing" step when working with an alum
hematoxylin in a standard H&E. Also keep in mind that improper fixation
can also contribute to poor nuclei staining.

	Jack

	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.

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