[Histonet] Re: Masson's trichrom - problem with nuclei staining
Itai Moshe
itai.moshe <@t> mail.huji.ac.il
Mon Sep 20 03:45:58 CDT 2010
Hi,
What concentration of HCL are you using in solution B ?
is it 1% in the end, so i will have to use 3ml of the standard 36% HCL in
100ml, not just 1ml as mentioned in the protocols ?
Itai
*gayle callis** **gayle.callis <@t> bresnan.net
*<histonet%40lists.utsouthwestern.edu?Subject=%5BHistonet%5D%20Re%3A%20Poor%20Weigerts%20Hematoxylin%20staining%20with%20Massons%0A%09Trichrome%20&In-Reply-To=>
*
**Fri Sep 17 11:40:13 CDT 2010*
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------------------------------
*In general, we found the original/classic Weigerts iron hematoxylin stain to
be weak and almost washed out of tissues after staining with Massons
Trichrome. We no longer use the original formula, but a more concentrated
modified formulation that is not differentiated out as much by the acidifed
solutions found in Mass Tri. We also found this problem with Massons
Trichrome kit components, where companies probably package the original
method's solutions.
We make up Weigerts fresh each time, and if it will last for a week for you,
fine......... but our work tends to be a one time staining with Mass Tri,
then weeks before it was done again. The problem is: ferric chloride
continues to oxidize the hematoxylin throughout over time, that week or
longer, weakening the iron hematoxylin staining capabililty. This is
discussed in Sheehan and Hrapchak Theory and Practice of Histotechnology.
Acid decalcified bone presents even more of a challenge, since nuclei
(DNA/RNA) in cells are hydrolyzed by acid decalcifiers, compromising
staining of nuclei, a problem seen with routine H&E staining. Deanna was
correct on her assessment of this stain for best results.
This modified Weigerts Hematoxylin was published in J of Histotechnology in
a paper on Kreybergs stain on skin. The second Extra Strength Weigerts was
found on Histonet years ago and I have not tried the latter. I suggest you
see which one you prefer, as we use the first Modified Weigerts. Over the
years, the modified gave us far superior nuclear staining with Massons
Trichrome.
Weigert's Iron Hematoxyin MODIFIED (found in J of Histotechnology in a
method for Kreybergs stain on mouse skin).
Solution A.
2% Hematoxylin in 95% ethanol
Solution B.
62% Ferric Chloride 4 ml
Distilled water 95 ml
Hydrochloric acid 1 ml
Mix equal amounts of Solution A and Solution B
MIX FRESH JUST BEFORE USE AND DISCARD AFTER USE.
Extra Strength Weigerts Iron Hematoxylin from Histonet (reference unknown,
but supposedly from J of Histotechnology and method originated by Mabel
Myli, Mayo Clinic)
Solution A: Hematoxylin 10 g
95% ethanol 100 ml
Solution B: 11.6 g Ferric Chloride
980 ml Distilled water
10 ml 25% hydrochloric acid
Working Stain Solution
Solution A 5 ml
Solution B 25 ml
Absolute Ethanol 20 ml
Staining time for both of these formulations is 10 minutes, followed by
rinsing for 10 min in running tap water (hematoxylin will be black)
Gayle M. Callis
HTL/HT/MT(ASCP)
************************
You Wrote:
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)
*
*2010/9/18 Andrea Marion **<amario3 <@t> uic.edu>**
*
>
> *Hi Itai,
>
> I am thinking that we will purchase a different Weigert's from another
> company, or try one of the modified ones that were recommended on the
> listserv.
>
> Running tap water only means that you are replacing the water the slides
> are sitting in continuously, so that they have a constant supply of fresh
> water. This removes any excess stain that may be bleeding from the slide,
> and prevents it from recoloring the water, and restaining the tissue.
> Essentially it helps pull more excess stain out of the slide. Leaving it
> in tap water does not have the same effect, because the dye will reach an
> equilibrium point whereby no more will be removed from the tissue. We use
> a coplin jar that we stain the slides in, and simply move it to the sink
> and let water run into the jar gently. You can achieve the same thing if
> you do not have a sink of some sort by constantly dumping out the water
> the slides are in, and replacing with clean water. It doesn't require any
> special equipment, just a source of running water and whatever container
> you normally stain slides in.
> **
> Andrea*
* *
***2010/9/18 Madary, Joseph **<MadaryJ <@t> medimmune.com>*
*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)*
>
*
*
*2010/9/18 Jack Ratliff **<ratliffjack <@t> hotmail.com>*
> *Dorn and Hart Microedge is now selling a Weigert's Hematoxylin kit. Maybe
> you could try it out and see if your results improve.
>
> **www.dornandhart.com* <http://www.dornandhart.com/>*
> **
> Jack*
*
*
*On Sat, September 18, 2010 9:03 am, Itai Moshe wrote:
> 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** *
*2010/9/18 gayle callis **<gayle.callis <@t> bresnan.net>**
*
>
> *You cannot use methyl green with Massons. Just don't use that Weigerts
> from
> the kit, make it up in house, simple. Ferric chloride goes into solution
> rapidly. Then use all the other kit components. I suggest you buy
> separate
> kit components from Newcomer Supply or Poly Scientific. I don't know where
> you are located for ordering??? You didn't say.
>
> Just to let you know, Massons IS NOT SPECIFIC FOR COLLAGEN, but stains all
> connective tissues in a section. If you want a stain specific for
> collagen,
> use van Giesons. That also uses Weigerts iron hematoxylin. The sections
> must be blotted after coming out of the van Giesons stain, or it washes out
> in alcohols. You then blot, air dry and cover slip - although you can dip
> in to a clearing agent to make the mounting media flow. Sirius red is whole
> different staining method for collagen, and final viewing of that should be
> with polarized light also.
>
> Also, collagen is very birefringent, polarized light examination even with
> an H&E works.
>
> When you do Weigerts hemtoxylin, the running tap water wash is important,
> or
> you will have a mess. Find a sink
>
> The problem with Weigerts and Massons, is as I explained before. All the
> other stains used in this method are acidified, also use phosphotungstic
> acid with phosphomolybdic acid, so there is a lot of acids to differentiate
> out the Weigerts hematoxylin, that is why the stronger formula for Weigerts
> works.
>
> There is no bluing with this hematoxylin, it is not that kind of
> hematoxylin, as the end result is black. BUT you must wash with tap water
> for at least 10 minutes to achieve correct staining. Immersion will NOT do
> the job. You would be standing there changing the water constantly.
> agitating for 10 minutes in many changes of tap water. Isn't there a sink
> located somewhere in your facility you can use for the hematoxylin portion
> of this staining? You can carry the slides back to finish the rest of the
> staining in a lab that doesn't have a sink.
>
> Methyl green will not work with Massons, nor any other collagen stain, it
> will wash out.
>
> Gayle Callis
> *
> *
> -----Original Message-----
> From: * *histonet-bounces <@t> lists.utsouthwestern.edu*<histonet-bounces <@t> lists.utsouthwestern.edu>
> *
> [mailto:**histonet-bounces <@t> lists.utsouthwestern.edu*<histonet-bounces <@t> lists.utsouthwestern.edu>
> *] On Behalf Of Itai Moshe
> Sent: Saturday, September 18, 2010 8:03 AM
> To: **deannal78 <@t> verizon.net* <deannal78 <@t> verizon.net>*; **amario3 <@t> uic.edu*<amario3 <@t> uic.edu>
> *;
> * *histonet <@t> lists.utsouthwestern.edu* <histonet <@t> lists.utsouthwestern.edu>*;
> **MadaryJ <@t> medimmune.com* <MadaryJ <@t> medimmune.com>*; **plott <@t> uab.edu*<plott <@t> uab.edu>
> *;
> * *atliffjack <@t> hotmail.com* <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* <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* <amario3 <@t> uic.edu>*>
> >
> > *To:* **histonet <@t> lists.utsouthwestern.edu*<histonet <@t> lists.utsouthwestern.edu>
> *
> > *Cc:* **itai.moshe <@t> mail.huji.ac.il* <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*<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* <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*<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* <http://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>
> *
> > <**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
> *
>
*
*
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