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

Sat Sep 18 11:34:42 CDT 2010

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

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
>
> 2010/9/17 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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>