[Histonet] O.C.T. what MW PVA and PEG?

[Tyrone Genade via Histonet]

Hello,

I'm using a cryoprotection protocol that involves 3-stage cryoprotection of 15% 
glucose O/N, then 30% + 50% OCT O/N and then finally O/N in OCT. Compared to 
previous protocols this works very well -- even when cutting through eyes and 
lenses (which had previously given a lot of grief).

My issue is that preparing the 30% + 50% OCT is a schlep. The OCT puts up a lot 
of resistance against mixing with the 60% sucrose. It would be much simpler if 
I could prepare 30% sucrose with powdered PVA and PEG. Does anyone know what MW 
polymers of PVA and PEG to use and what concentrations to approximate 
commercial (Scigen Tissue-Plus) OCT?

Thanks

Tyrone Genade Ph.D.
Quillen College of Medicine
ETSU
Johnson City, TN
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Re: [Histonet] Water under sections

[Tyrone Genade via Histonet]

Hello,


On Saturday, January 23, 2021 1:00 PM, 
 wrote:

> In my lab, water under the sections is unique to charged slides.
> And you
> are correct, if there is water under the section when the slides
> are heated
> for antigen retrieval, the boiling (or at least very hot) water
> will damage
> or entirely destroy the section.

I had this very problem and the simple solution was to bake the slides 
overnight at 56oC (melting temp of the wax I was working with). I would still 
heat on a hot plate before dewaxing. Baking at 45oC also seemed to work. 
Whether this would work for you would depend on the sensitivity of your 
epitopes to prolonged heat.

Good luck... every now and then a section would still detach during antigen 
retrieval.

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[Histonet] stain to demonstrate silver ions in tissue?

[Tyrone Genade via Histonet]

Hello,

A colleague wants to determine the location and relative abundance of
silver impregnation in tissue samples (skin). Is there an histological
method for this?

I know silver will stain the tissue all by itself, but it is important we
are able to identify the limit of the penetration of the silver, which will
likely be greater than we could detect by eye from silver's natural
tendency to react with protein.

Silver apparently slightly enhances the staining with DAB. My experience
with Ni/Co intensification is that this metal catalyzed process happens
without the need for HRP. So after blocking the silver + H2O2 should cause
the DAB to precipitate locally at sites of Ag accumulation. Any thoughts on
this idea?

Thanks
-- 
Tyrone Genade
Johnson City, Tennessee
tel: (+1) 712 230 4101

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Re: [Histonet] Chromic Acid Disposal

[Tyrone Genade via Histonet]

Hello,

For the disposal of Chromic acid see
https://www.chemistry.nus.edu.sg/PSSO/safety/Special%20Chemical%20Waste.htm
 and
https://study.com/academy/lesson/chromic-acid-solution-preparation-disposal-hazards.html
(more
detailed).

You can precipitate the chrome as Cr(III) and store this solid for waste
disposal instead of liquid chromic acid. In the process the acid is
neutralized and the supernatant (of sulfates, potassium and sodium ions)
diluted and washed down the sink.

I hope that helps.

Tyrone
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Re: [Histonet] Histonet Digest, Vol 180, Issue 7

[Tyrone Genade via Histonet]

Hello,

An update on my question:

On Fri, Nov 9, 2018 at 12:22 PM 
wrote:

>
> Message: 1
> Date: Thu, 8 Nov 2018 15:45:35 -0600
> From: Tyrone Genade 
> To: histonet 
> Subject: [Histonet] counter stains for Sudan Black B
> Message-ID:
> <
> caeyee3nxb7nwezrbl_uodtntjtqidhryxksdmgv4kvmv_pq...@mail.gmail.com>
> Content-Type: text/plain; charset="UTF-8"
>
> Hello,
>
> I need a counterstain for use together with Sudan Black B. I am using this
> method: https://www.ncbi.nlm.nih.gov/pubmed/27812872 which uses Nuclear
> Fast Red. I don't have nuclear fast red... In an updated protocol by these
> authors they use hematoxylin but in a previous trial I found it was too
> dark and make visualizing the Sudan Black signal too difficult. I'm using
> Sigma's Mayer's Hematoxylin solution. It is past its sell-by date and just
> won't blue nicely any more..
>
> I have available Methyl Green, Neutral Red and  Safranin  O. The former
> washes out in aqueous/glycerol mounting medium (required for the protocol).
> How well does Neutral Red and Safranin work with an aqueous mounting
> medium?
>

There were no replies but Neutral Red worked.

Does anyone know of a paper describing the cellular structures stained by
Neutral red? https://en.wikipedia.org/wiki/Neutral_red states that is
stains the Golgi apparatus and Nissl bodies and that living cells take up
the stain in lysosomes.
http://stainsfile.info/StainsFile/stain/nuclei/neutred.htm says it stains
the nucleus. I see a darker stained red halo around the nucleus and the
nucleus is stained reddish. It also seems to stain the aggresome, that is
it stains small structures juxtaposed to the nucleus. The Sudan Black seems
to overshadow the stain. Any speculation what is going on? Is the neutral
red staining structures that would also contain lipofuscin?

Thanks
-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

Romans 6:23: The gift of God is eternal life through Christ Jesus our Lord.
To find out how to receive this FREE gift visit http://www.alpha.org.

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[Histonet] counter stains for Sudan Black B

[Tyrone Genade via Histonet]

Hello,

I need a counterstain for use together with Sudan Black B. I am using this
method: https://www.ncbi.nlm.nih.gov/pubmed/27812872 which uses Nuclear
Fast Red. I don't have nuclear fast red... In an updated protocol by these
authors they use hematoxylin but in a previous trial I found it was too
dark and make visualizing the Sudan Black signal too difficult. I'm using
Sigma's Mayer's Hematoxylin solution. It is past its sell-by date and just
won't blue nicely any more..

I have available Methyl Green, Neutral Red and  Safranin  O. The former
washes out in aqueous/glycerol mounting medium (required for the protocol).
How well does Neutral Red and Safranin work with an aqueous mounting medium?

Thanks
-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

Romans 6:23: The gift of God is eternal life through Christ Jesus our Lord.
To find out how to receive this FREE gift visit http://www.alpha.org.

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Re: [Histonet] Histonet Digest, Vol 171, Issue 4

[Tyrone Genade via Histonet]

Hello all, and especially those that offered advice.

Thanks for the advice. I will be experimenting with the options today and
tomorrow.

Thanks for being so generous with information.

Kind regards

On Sun, Feb 4, 2018 at 12:00 PM, 
wrote:

> Date: Sat, 3 Feb 2018 13:50:11 -0500
> From: Bob Richmond 
>
> Bryan Llewellyn refers you to
> http://stainsfile.info/StainsFile/stain/oversight/romanowsky.htm
>
> That method prescribes neutral buffered formalin fixation. Us old-timers
> preferred Zenker fixation, which you can't do today on account of the
> mercury in it. You may have to try different fixatives.
>
> The differentiation under microscopic control is really necessary - when I
> was a resident at Cornell Medical Center in NYC fifty years ago, the
> histotechs would push the residents off the double-headed microscopes when
> they sat down to differentiate the bone marrow biopsy section Giemsa
> stains.
>
> The old-timers used a 10% solution of colophonium rosin (almost pure
> abietic acid) in alcohol, instead of acetic acid, to differentiate the
> stain.
>
> You buy Giemsa or Wright or Romanovsky stain already dissolved in absolute
> alcohol. You have to be a fanatic about not contaminating your stock bottle
> with the tiniest trace of water.
>
> Bob Richmond
> Samurai Pathologist
> Maryville TN
>


-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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[Histonet] Wright-Giemsa for sections?

[Tyrone Genade via Histonet]

Hello,

Can the Wright-Giemsa stain be used on fixed, paraffin embedded sections?
Does anyone have a protocol?

I want to examine hematopoietic tissue of fish, i.e. the head kidney. No
smears or imprint possible. I would like to use Wrights so I can use the
same stain for blood smears.

Thanks

-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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Re: [Histonet] Disposal of Bouin's Solution

[Tyrone Genade via Histonet]

Hello,

Regarding disposal of Bouin's solution the following protocol might be
helpful/amusing/terrifying:
https://phc.amedd.army.mil/PHC%20Resource%20Library/Bouins_fixative_solution_FS%20_37-007-0913.pdf


For IHC the argument is that Bouin's doesn't destroy antigens as compared
to other fixatives but I think this is a problem specific to certain
antigens rather than a general issue---and those antigens can be preserved
by other means now. It also seems to preserve nuclei much better.

Bye
-- 
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Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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[Histonet] FYI: FastRed vs 88% formic acid

[Tyrone Genade via Histonet]

Hello,

Sticking with the theme...

In case you are curious Abcam's FastRed (
http://www.abcam.com/liquid-fast-red-substrate-kit-ab64254.html) on IHC
sections is resistant to 88% formic acid. There is a little fading but it
holds up well.

Bye
-- 
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Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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[Histonet] FYI: DAB vs 88% formic acid

[Tyrone Genade via Histonet]

Hello,

In case you are curious DAB on IHC sections is resistant to 88% formic acid.

If you want to know why I would put a DAB stained section in formic acid, I
have conformation specific antibody that I want to use in a
double-labelling experiment using an antibody needing formic acid epitope
retrieval...

Incidentally, having trouble matching antibodies for double labelling? This
might be useful: Antibody Elution Method for Multiple Immunohistochemistry
on Primary Antibodies Raised in the Same Species and of the Same Subtype
http://journals.sagepub.com/doi/10.1369/jhc.2009.953240 I haven't tried
this method yet but I will...

bye
-- 
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Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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[Histonet] Maximow Method

[Tyrone Genade via Histonet]

Hello,

I was reading a book from the 60s on the anatomy of aging in man and
animals and the author mentioned using a hematoxylin-eosin Y-Azure-II stain
to show the lymphocytes. Some searching came up with the Maximow Method.
The online protocol I found (for bone marrow):
https://emsdiasum.com/microscopy/technical/datasheet/26252.aspx mentions
the use of Zenker’s or Formalin.

In another old book, Putt's Manual of Histochemical Staining Methods, the
authors says 10% fomalin, Helly's or Zenker's fluid for fixation. I am
definitely not going to start using Zenker's (I get enough grief from the
H officer about picric acid). I normally use Davidson's fixative to fix
and decalsify my fish (formaldehyde, acetic acid + ethanol). Anyone know if
this staining method is compatible with Davidson's fixative?

Would this eosin-Y solution be suitable:
http://www.sigmaaldrich.com/catalog/product/sigma/ht110316?lang=en=US
 for preparing the Eosin-Y Azure-II working solution?

Thanks
-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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[Histonet] head kidney issues

[Tyrone Genade via Histonet]

Hello,

I am doing IHC on fish tissues, in particular the head kidney.

I am using DAB as chromogen. The tissue was fixed in Davidson's Fixative
and processed for wax. Sections were dried in an oven overnight and then
dewaxed and citrate was used for heat antigen retrieval. The sections were
then permeablized with PBS-Triton and incubated in methanol with H202 for
30 min followed by 20 minutes in pH 2.3 citrate (biochemically speaking it
should all be citric acid at this point) to denature any surviving
endogenous peroxidase. The last step may seem a bit extreme but I was still
getting signs of endogenous peroxidase activity in the head kidney sections
after the methanol and still am. Now I wonder if it is something else?

I found http://link.springer.com/article/10.1007/BF01003535 and saw this
http://www.ncbi.nlm.nih.gov/pubmed/16242344 and now wonder if the issue
isn't  5′-nucleotidase. The strong signal I am seeing has a granular
appearance (macrophages?). Has anyone had similar experiences and found a
solution? The former article implies using EDTA as an inhibitor.

Thanks
-- 
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Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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[Histonet] metal enhanced DAB

[Tyrone Genade via Histonet]

Hello,

I'm using the Co/Ni enhanced DAB from Histological & Histochemical Methods
(2nd Ed). Does the solution normally turn a blue-gray after adding the
H2O2? I am getting a lot of back ground and precipitate on the sections.
The pH is 7.3.

I had used a similar method for Western Blotting that took 5 minutes for
the solution to change color. This is a near instantaneous color change.

Thanks

Tyrone
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Re: [Histonet] Davidson's fixative and IHC question

[Tyrone Genade via Histonet]

Hello,

I am using Davidson's fixative to fix whole fish and I am very happy with
the results. I had tried 4% PFA but had issues with collapsed eyes and
detached retinas... No such issues with Davidson's or Bouin's. In my
experience I get sharper nuclear staining by H and better declacification
with Davidson's. The integrity of the brain tissues is good with less
shrinkage than with Bouin's.

I fix whole fish over night and then move them to 70% ethanol.

I had issues with autofluoresence with Davidson's (no surprise there) but
it was easily remedied using Gayle Callis' glycine protocol (available
somewhere on histonet). Same story for Bouin's.

Some antigens are lost and seem irretrievable.  The anti-GFAP antibody,
GA5*, worked fine on PFA fixed fish but the epitope couldn't be retrieved
in Bouin's fixed fish. I'm not sure about Davidson's but using heat induced
citric acid antigen retrieval (pH 5, 80 min at 60 oC) has worked for
several epitopes.

* If memory serves, this is a phosphate-epitope so that could be something
to look out for.

I would advise testing several different fixatives and seeing which works
the best for your application. If you are working with soft tissue, such as
brain, a zinc fixative might be better for the preservation of delicate
epitopes: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1919503/ .

Bye

Tyrone
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Re: [Histonet] microtome issues: thick and no sections (Tyrone Genade)

[Tyrone Genade via Histonet]

Hello,

Thanks to all who responded to my question. The solution, it seems was very
simple.

Several people reported similar experiences and issues with the specimen
clamp not fitting tightly and that I should clean the clamp or block. My
blocks are clean and I thought the clamp was too... but when I ran a set of
forceps along the inner, upper edge of the clamp a tiny blob of wax was
found. A test block cut normally.

I'm amazed a tiny piece of wax could cause so much  trouble!

Thanks to everyone who offered help.

Bye
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[Histonet] microtome issues: thick and no sections

[Tyrone Genade via Histonet]

Hello,

I am having a problem with sectioning.

I'm cutting 5 um sections but with the turn of the microtome I will miss
the block, or take a small piece of the block/tissue and then the next
section ends up thick. This missing can take several turns of the wheel so
I can get really big sections. I have checked the angle of the knife to the
block and the tension along the blade but these are fine. I worry that the
issue might be temperature. The microtome is on a bench against an outside
wall. The temperature is about 15 oC. Could this be the issue?

I am not cooling the block on ice. Previous experience with block-cooling
was that I would get thick and thin sections that way... this is a very
different problem.

Thanks

-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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Re: [Histonet] Histonet Digest, Vol 145, Issue 24

[Tyrone Genade via Histonet]

Hello

From: "Dube, Emily" 
> Subject: [Histonet] fungus growing on paraffin blocks
>
> I work for a biobanking research study and recently we discovered some of
> our paraffin blocks had mold/fungus growing on the tissue. We were advised
> to soak the blocks in bleach. Does anyone else have any suggestions on what
> to do to ensure the fungus is eliminated without ruining the tissue?
>
> Emily
>

I doubt bleach alone will be sufficient.It may kill the fungus growing on
the outside but probably not penetrate deep enough to kill hyphae deeper in
the tissue. Even if it were successful spores in the current environment
could still regrow. You need to sterilize the storage area.

An azide soak might prove more useful as the residual azide would inhibit
further growth. Azide is very toxic so this would need to be taken in
account when the tissue is processed... Another idea would be to use
mothballs. The 1,4-dichlorobenzene has antifungal properties. I don't know
how this will effect future processing of the tissue. Bagging blocks in
plastic bags with mothballs might make the storage space more user friendly.

Merry Christmas!

-- 
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Orange City, Iowa
tel: (+1) 712 230 4101
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[Histonet] methyl green salt supplier

[Tyrone Genade via Histonet]

Hello,

Anyone know of a source for ethyl violet free methyl green powder? I see
Sigma has discontinued their M6776 powder.

Thanks

-- 
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Orange City, Iowa
tel: (+1) 712 230 4101
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[Histonet] positive control tissues needed: Parkinson's

[Tyrone Genade via Histonet]

Hello,

I am looking for positive  control tissues of Parkinson's Disease. I have
contacted National Disease
Research Interchange but they can only supply me with whole brains and
hemispheres which is way more than I need and don't want to waste tissue. I
am looking for samples of the following:
substantia nigra
Dorsal Motor Nucleus CN ten (vagus)
any tissue that will show alpha-synuclein Lewy neurites

Is there anyone out there who has a tissue block spare of the above that
they could donate towards my research?

Thanks
-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
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[Histonet] protocol for HE staining of Lewy Bodies

[Tyrone Genade via Histonet]

Hello,

I'm currently using HE to stain sections for Lewy bodies. I'm using Sigma
HE solutions (
http://www.sigmaaldrich.com/catalog/product/sigma/ht110316?lang=enregion=US
and
http://www.sigmaaldrich.com/catalog/product/sigma/mhs16?lang=enregion=US).
The sections are immersed in the solutions 5 min for H and 4 min for E. At
4 min the staining was very intense so we tried it at 1 min which gave nice
bright collagen and erythrocyte in my fish sections. (Tissues were fixed in
Davidson's Fluid and paraffin imbedded.) We are now going to try 2
minutes... The H is also very intense so I will try a shorter staining
period as well.

Thanks
-- 
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org

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[Histonet] are you desperate enough to hire a B.Sc. graduate?

[Tyrone Genade via Histonet]

Hello,

Last week a I visited Pittsburgh and had a chance to talk with a fellow
histologist there. He remarked on the dire state his lab is in with respect
to finding qualified histologists to employ; and that the lab was now
considering hiring B.Sc. graduates and training them up in sectioning
etc... Is this a common problem, the trouble finding qualified histotechs,
and are other labs also considering hiring B.Sc. graduates to staff their
labs?

I have one student working with me (with medical school aspirations) who
sections beautifully. There is surely talent out there...

Bye
-- 
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Orange City, Iowa
tel: (+1) 712 230 4101
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[Histonet] Re. Decalcification with formic acid sodium

[Tyrone Genade via Histonet]

It isn't very clear, but for the Formic Acid/sodium Citrate buffer you have
to use trisodium citrate. 10 g of disodium or monosodium citrate wouldn't
work as well...

--
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Orange City, Iowa
tel: (+1) 712 230 4101
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