[Histonet] modified gomori stain
Hi,
I need help with modified gomori stain. I cant get the muscle to be green. Could anyone help me.
Dr. Manal Galal
Consultant Pathology
Institute of Neuromotor Disabilities
Cairo, Egypt
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Today's Topics:
1. RE: Trichrome staining and fibrin
(Marshall Terry Dr, Consultant Histopathologist)
2. substrates (Perry, Margaret)
3. toe nails (Conlon, Paula F.)
4. Re: substrates (Rene J Buesa)
5. Re: toe nails (Rene J Buesa)
6. Re: toe nails (Chris Pomajzl)
7. ScyTek's serum free pro-block protein (Maria Mejia)
8. RE: toe nails (Anthony F. Boris)
9. Manual cover slip method - DiscoverSlip? (Geoffrey White)
10. Re: substrates (Susan Q Wells)
11. Sphingomyelin (mtitford@aol.com)
12. High profile blades (Snider, Deanna)
13. Mechanical testing-Dallas-Fort Worth (Ashwin Nair)
----------------------------------------------------------------------
Message: 1
Date: Wed, 14 Jun 2006 13:39:24 +0100
From: "Marshall Terry Dr, Consultant Histopathologist"
Subject: RE: [Histonet] Trichrome staining and fibrin
To: "Bryan Hewlett" , Timo V?is?nen
,
Message-ID:
Content-Type: text/plain; charset="iso-8859-1"
Well, that's telling me:-)
All these years, the success I've had with primary mercury fixation (I had the original article once), and lack of success with anything other was entirely due to something else, the techs washing too much in one instance and little enough in another.
Who would have thought it.
However it may explain my observation that "At times it works and at times not."
Actually, the only time you can be confident that you are looking at fibrin, is when it is in strands - easily recognised in H&E.
To call anything else fibrin on any tinctorial stain is something akin to a leap of faith.
But - I'll get you back re. your "bees can't fly" thing.
Take this/that: :-)
The "science has proved that bees can't fly" urban myth originated in a 1934 book by entomologist Antoine Magnan, who discussed a mathematical equation by Andre Sainte-Lague, an engineer. The equation proved that the maximum lift for an aircraft's wings could not be achieved at equivalent speeds of a bee. I.e., an airplane the size of a bee, moving as slowly as a bee, could not fly. Although this did not mean a bee can't fly (which after all does not have stationary wings like the posited teensy aircraft), nevertheless the idea that Magnan's book said bees oughtn't be able to fly began to spread.
Dr Terry L Marshall, B.A.(Law), M.B.,Ch.B.,F.R.C.Path
Consultant Pathologist
Rotherham General Hospital
South Yorkshire
England
terry.marshall@rothgen.nhs.uk
-----Original Message-----
From: Bryan Hewlett [mailto:bhewlett@cogeco.ca]
Sent: 13 June 2006 17:14
To: Marshall Terry Dr, Consultant Histopathologist; Timo Väisänen;
histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Trichrome staining and fibrin
Terry,
Actually NOT!
True, Lendrum's original papers(1962) recommended fixation in picro-mercuric
alcohol for 3 weeks (a tad unnecessary that!).
However, both Prof. Lendrum and Bill Slidders also successfully used
formaldehyde fixed material that was treated in Bouin immediately prior to
staining.
The key to all of the Masson Type trichromes on formaldehyde-fixed tissue,
is to use a pretreatment in picric acid.
This re-aligns the reactive side chains on the proteins, so that there is a
predominance of basic amino groups and hence maximal binding of anionic
dyes.
Mercury fixation is simply not necessary! Nor is Zinc substitution. (see
Troubleshooting Histology stains: Horobin and Bancroft 1998, page 129)
The lack of consistency, in obtaining good red colour, is due variability in
the staining procedure (the post dye water rinses), NOT the fixation.
Once this is addressed, the inconsistency goes away.
That is true for the original Masson, Picro-Mallory (all variants), MSB and
Masson 44/41.
Achieving consistent good red colour with formaldehyde fixed material is
easy, if the staining mechanisms are understood and those unnecessary water
rinses removed.
Been doing it for over 40 years!
Bryan
(Engineers can prove that the bumble bee simply cannot fly. However, the
bumble bee doesn't know that and flies anyway)
----- Original Message -----
From: "Marshall Terry Dr, Consultant Histopathologist"
To: "Bryan Hewlett" ; "Timo Väisänen"
;
Sent: Tuesday, June 13, 2006 11:27 AM
Subject: RE: [Histonet] Trichrome staining and fibrin
Bryan,
You know full well that MSB required initial staining in mercury. I know
that zinc is an adequate replacement.
Attempting to get a consistent good red colour with formalin fixed material
is futile, as is post-fixation.
At times it works and at times not.
IMO, Masson is useless for fibrin.
Dr Terry L Marshall, B.A.(Law), M.B.,Ch.B.,F.R.C.Path
Consultant Pathologist
Rotherham General Hospital
South Yorkshire
England
terry.marshall@rothgen.nhs.uk
-----Original Message-----
From: Bryan Hewlett [mailto:bhewlett@cogeco.ca]
Sent: 13 June 2006 15:52
To: Timo Väisänen; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Trichrome staining and fibrin
Timo,
Try the Lendrum MSB trichrome variant, it was designed to demonstrate
fibrin.
I will send the method via a separate e-mail.
Bryan
----- Original Message -----
From: "Timo Väisänen"
To:
Sent: Tuesday, June 13, 2006 10:33 AM
Subject: [Histonet] Trichrome staining and fibrin
> Hello all,
>
> I have struggled with getting Masson Trichrome (Goldner) staining to work
> with
> kidney biopsies. The problem is that our pathologist does not get positive
> fibrin staining to glomeruli in diseased kidneys. Those areas of the
> glomeruli
> that should contain fibrin, at least according to the pathologist, are
> green
> (Lichtgrun) and not red, as they should be, whatever I do. I have tried to
> enhance the staining with Bouin's fixative (+56C) pretreatment but it did
> not
> change the overall situation. However, the red stain was more intense in
> skin
> samples containing fibrin (formalin fixed). I have also tried another
> Trichrome
> staining with Crocein Scarlet 7B without any luck. In our lab kidney
> biopsies
> are routinely fixed with alcoholic Bouin's. As far as I know it should be
> compatible with the Masson protocol we use. Any ideas? Any good protocols
> that
> I could try?
>
> Thank's in advance,
>
> Timo
>
>
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> Histonet@lists.utsouthwestern.edu
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>
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Message: 2
Date: Wed, 14 Jun 2006 09:08:36 -0500
From: "Perry, Margaret"
Subject: [Histonet] substrates
To:
Message-ID:
Content-Type: text/plain; charset="us-ascii"
We are trying to do double staining and are looking for two different
colors of substrate and we can't use DAB due to melanin. Does anyone
know of a green stain that is xylene compatible? Vector has Nova Red
and a blue color but we use hematoxylin as a counterstain. It would
mean an extra step for us to counterstain in Methyl Green
We use a DAKO autostainer.
Margaret Perry HT(ASCP)
South Dakota State University
Animal Research and Diagnostic Lab
Brookings SD 57007
Margaret.Perry@sdstate.edu
------------------------------
Message: 3
Date: Wed, 14 Jun 2006 10:16:10 -0400
From: "Conlon, Paula F."
Subject: [Histonet] toe nails
To: "Histonet \(E-mail\)"
Message-ID:
Content-Type: text/plain; charset="iso-8859-1"
Hi Histonetters, does anyone have a good procedure for sectioning nails and keeping them on the slides during h&e staining and pas staining?Most of the time our sections wash off. Thanks for your help.
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------------------------------
Message: 4
Date: Wed, 14 Jun 2006 07:23:19 -0700 (PDT)
From: Rene J Buesa
Subject: Re: [Histonet] substrates
To: "Perry, Margaret" ,
histonet@lists.utsouthwestern.edu
Message-ID: <20060614142319.7044.qmail@web61216.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Margaret:
Confronted with that situation I would eliminate the melanine better. Oxidize it with 0.1% potassium permanganate for 2 hours; wash and remove the oxidized melanine with 0.3% oxalic acid followed by washing. The section should be free of melanine pigment/colour afterwards and you can use DAB (for brown) and DAB + Ni salts for dark blue as double substrates.
Hope this will help you!
René J.
"Perry, Margaret" wrote:
We are trying to do double staining and are looking for two different
colors of substrate and we can't use DAB due to melanin. Does anyone
know of a green stain that is xylene compatible? Vector has Nova Red
and a blue color but we use hematoxylin as a counterstain. It would
mean an extra step for us to counterstain in Methyl Green
We use a DAKO autostainer.
Margaret Perry HT(ASCP)
South Dakota State University
Animal Research and Diagnostic Lab
Brookings SD 57007
Margaret.Perry@sdstate.edu
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Message: 5
Date: Wed, 14 Jun 2006 07:25:13 -0700 (PDT)
From: Rene J Buesa
Subject: Re: [Histonet] toe nails
To: "Conlon, Paula F."
, "Histonet
\(E-mail\)"
Message-ID: <20060614142513.15660.qmail@web61219.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Paula:
I am forwarding privately a summary on Toenails I prepared recently that I hope will answer your questions.
René J.
"Conlon, Paula F."
wrote:
Hi Histonetters, does anyone have a good procedure for sectioning nails and keeping them on the slides during h&e staining and pas staining?Most of the time our sections wash off. Thanks for your help.
See our web page at http://www.lahey.org for a full directory of Lahey sites, staff, services and career opportunities.
THIS MESSAGE IS INTENDED FOR THE USE OF THE PERSON TO WHOM IT IS ADDRESSED. IT MAY CONTAIN INFORMATION THAT IS PRIVILEGED, CONFIDENTIAL AND EXEMPT FROM DISCLOSURE UNDER APPLICABLE LAW. If you are not the intended recipient, your use of this message for any purpose is strictly prohibited. If you have received this communication in error, please delete the message and notify the sender so that we may correct our records.
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Message: 6
Date: Wed, 14 Jun 2006 09:41:48 -0500
From: "Chris Pomajzl"
Subject: Re: [Histonet] toe nails
To: "HISTONET"
Message-ID: <002e01c68fc0$b2fb6330$26fca8c0@CSP>
Content-Type: text/plain; charset="iso-8859-1"
Good question. We have dealt with this issue for many years, and we have
come up with a solution that has worked for some time.
First of all, sections are picked up on "+" coated slides that have been
smeared with egg albumin. We separate eggs from the grocery store and keep a
stock of the egg white albumin in a bottle. Maybe 100ml at a time, keep it
refrigerated. When cutting nails, put 2-3 drops of albumin on the slide and
smear to cover all of the glass.
We cut 3 sections of the nail specimen. 1 for H&E, 1 for PAS, and 1 extra
for back-up. We bake the slides at 55'C overnight, and then at 70'C for 20
minutes prior to staining the following day.
This seems to work very well for us.
----- Original Message -----
From: "Conlon, Paula F."
To: "Histonet (E-mail)"
Sent: Wednesday, June 14, 2006 9:16 AM
Subject: [Histonet] toe nails
Hi Histonetters, does anyone have a good procedure for sectioning nails and
keeping them on the slides during h&e staining and pas staining?Most of the
time our sections wash off. Thanks for your help.
See our web page at http://www.lahey.org for a full directory of Lahey
sites, staff, services and career opportunities.
THIS MESSAGE IS INTENDED FOR THE USE OF THE PERSON TO WHOM IT IS ADDRESSED.
IT MAY CONTAIN INFORMATION THAT IS PRIVILEGED, CONFIDENTIAL AND EXEMPT FROM
DISCLOSURE UNDER APPLICABLE LAW. If you are not the intended recipient, your
use of this message for any purpose is strictly prohibited. If you have
received this communication in error, please delete the message and notify
the sender so that we may correct our records.
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------------------------------
Message: 7
Date: Wed, 14 Jun 2006 08:11:41 -0700
From: Maria Mejia
Subject: [Histonet] ScyTek's serum free pro-block protein
To: histonet@lists.utsouthwestern.edu
Message-ID:
Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed
To anyone who using ScyTek's serum free pro-block protein, please let
me know
what you think of it. In IHC, do you (only) use it as a diluent for
the antibodies or also
use it in the washes? Any information you can provide will be greatly
appreciated.
Yours
Maria Bartola Mejia
UCSF
Depart. of Neurosurgery
San Francisco, CA 94103
------------------------------
Message: 8
Date: Wed, 14 Jun 2006 11:51:21 -0400
From: "Anthony F. Boris"
Subject: RE: [Histonet] toe nails
To: "HISTONET"
Message-ID:
Content-Type: text/plain; charset="utf-8"
We use charged slides from Premiere. keep in 65 degree oven for 30 minutes and stain. We have not had any fall off since we switched to these slides. When cutting you can use a 5% solution of KOh for a "surface decal". This breaks down the keratin a little bit (same stuff as in NAIR). Helps to get decent sectioning.
Tony
-----Original Message-----
From: Chris Pomajzl [mailto:cpomajzl@cpllabs.com]
Sent: Wed 6/14/2006 10:41 AM
To: HISTONET
Cc:
Subject: Re: [Histonet] toe nails
Good question. We have dealt with this issue for many years, and we have
come up with a solution that has worked for some time.
First of all, sections are picked up on "+" coated slides that have been
smeared with egg albumin. We separate eggs from the grocery store and keep a
stock of the egg white albumin in a bottle. Maybe 100ml at a time, keep it
refrigerated. When cutting nails, put 2-3 drops of albumin on the slide and
smear to cover all of the glass.
We cut 3 sections of the nail specimen. 1 for H&E, 1 for PAS, and 1 extra
for back-up. We bake the slides at 55'C overnight, and then at 70'C for 20
minutes prior to staining the following day.
This seems to work very well for us.
----- Original Message -----
From: "Conlon, Paula F."
To: "Histonet (E-mail)"
Sent: Wednesday, June 14, 2006 9:16 AM
Subject: [Histonet] toe nails
Hi Histonetters, does anyone have a good procedure for sectioning nails and
keeping them on the slides during h&e staining and pas staining?Most of the
time our sections wash off. Thanks for your help.
See our web page at http://www.lahey.org for a full directory of Lahey
sites, staff, services and career opportunities.
THIS MESSAGE IS INTENDED FOR THE USE OF THE PERSON TO WHOM IT IS ADDRESSED.
IT MAY CONTAIN INFORMATION THAT IS PRIVILEGED, CONFIDENTIAL AND EXEMPT FROM
DISCLOSURE UNDER APPLICABLE LAW. If you are not the intended recipient, your
use of this message for any purpose is strictly prohibited. If you have
received this communication in error, please delete the message and notify
the sender so that we may correct our records.
_______________________________________________
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------------------------------
Message: 9
Date: Wed, 14 Jun 2006 08:52:10 -0700 (PDT)
From: Geoffrey White
Subject: [Histonet] Manual cover slip method - DiscoverSlip?
To: histonet@lists.utsouthwestern.edu
Message-ID: <20060614155210.68428.qmail@web36513.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Dear all,
at the time our research group is looking for new manual method for cover our slides during the hybridization. In the past we used standard glass cover slips but we are not really satisfied with this method.
Just I saw at Biocompare a new method with the name DiscoverSlip. This technology looks very interesting.
Have any work with DiscoverSlip or saw this technology working?
Thanks
Geoffrey
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Message: 10
Date: Wed, 14 Jun 2006 12:01:42 -0400
From: Susan Q Wells
Subject: Re: [Histonet] substrates
To: Rene J Buesa
Cc: histonet@lists.utsouthwestern.edu, "Perry, Margaret"
Message-ID: <449032E6.8030300@bms.com>
Content-Type: text/plain; format=flowed; charset=ISO-8859-1
You may lose your epitopes with this procedure, if so 10% H202 in PBS
for 30 minutes may lighten the melanin pigment enough
to view your chromagen. I recently used Vulcan Fast Red and Bajoran
Purple from Biocare Medical with great success where melanin pigment was
present.
Sue Wells HT(ASCP), QIHC
Rene J Buesa wrote:
>Margaret:
> Confronted with that situation I would eliminate the melanine better. Oxidize it with 0.1% potassium permanganate for 2 hours; wash and remove the oxidized melanine with 0.3% oxalic acid followed by washing. The section should be free of melanine pigment/colour afterwards and you can use DAB (for brown) and DAB + Ni salts for dark blue as double substrates.
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