[Histonet] Double stain IHC question

[Steven Weston via Histonet]

This is probably most easily done using immunofluorescence since it works well 
on frozen tissue. You could use a streptavidin linked FITC tag for the human 
biotinylated Ab, and then use a red tagged anti IgG directed against the 
species of the other antibody. (hoescht reagent will stain the nuclei)

If however you want to use immunohistochemistry you can use a strepatvidin 
linked alkaline phosphatase with BCIP/NBT (blue chomogen) for the biotinylated 
Ab and a polymer based peroxidase secondary for the other antibody and 
visualise using DAB. For nuclear staining you can use nuclear fast red. I have 
used this method recently to stain cytospots successfully for similar 
macrophage markers (inos and cd68).

Hope this helps.

regards

steve weston
lab manager
Breathe-Well CRE
UTAS-SOM


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Re: [Histonet] CPT Code 88399

[S hay via Histonet]

Please post answers to the group. We would like to see the responses as
well.
On Feb 19, 2016 2:04 PM, "Amy Self via Histonet" <
histonet@lists.utsouthwestern.edu> wrote:

> Happy Friday and I hope everyone has a great weekend,
>
> Can anyone tell me what the CPT code 88399 can be used for in pathology?
>
> We have been using this code to track mail-outs for productivity purposes
> but was told today that because there is not a dollar amount attached to
> this code that we could no longer use it.
>
> Is there a cpt code that can be used for when sending pathology material
> out to other facilities when the patient is being seen by a physician for
> continued healthcare?
>
> Thanks in advance for you help,
> Amy Self
> Histology Lab Senior Tech
> Lab
> Tidelands Georgetown Memorial Hospital
> 606 Black River Road
> Georgetown, SC 29440
> 843-520-8711
> as...@tidelandshealth.org
>
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Re: [Histonet] Double stain IHC question

[Ray via Histonet]

All, 
I guess I'm reading (or misreading) the situation differently than some.  That 
this is frozen, and not FFPE, is given originally. 
  
But I'm reading this as two different antibodies marking the very same protein. 
[Antibody=same protein A].  Is the original question can you mark same protein 
with two different antibodies to it?  I have done this with two antibodies to 
DIFFERENT epitopes of the same protein from home grown antibodies and we knew 
which antibody was directed to which epitope.  If two different antibodies are 
directed to the SAME epitope on the same protein they will just compete and 
favor that one with greater affinity/avidity/more conducive physical 
characteristics for binding.  Then colocalizing with fluorochromes is certainly 
possible as is done routinely. 
  
Ray (now in Spokane WA) 

- Original Message -

From: "Caroline Miller via Histonet"  
To: "gu lang"  
Cc: "Histonet@Lists. Edu"  
Sent: Saturday, February 20, 2016 8:35:18 AM 
Subject: Re: [Histonet] Double stain IHC question 

+ to Gudrun's comments. 

My addition is that fluorescence labels may work for this application. 
Again, depending on the species of the antibodies. The other advantage of 
the fluorescence is that you would be able to truly see the colocalization 
(yellow for instance if you used red and green fluorescent antibodies). 
Rather than the muddiness I have often got when trying two antibodies in 
bright field chromagenic stains. 

Remember though autofluorescence is greatly increased with paraffin 
tissues, that may put another issue on your plate. 

Good luck! 

yours, 
mills 

On Sat, Feb 20, 2016 at 3:47 AM, Gudrun Lang via Histonet < 
histonet@lists.utsouthwestern.edu> wrote: 

> In my opinion, this would only be possible, if the commercial and the 
> homegrown antibody are from different species. For example one from mouse 
> and one from rabbit. 
> Then you can proceed with different secondaries (goat anti mouse conjugated 
> with peroxidase, goat anti rabbit conjugated with alkaline phosphatase). 
> Then chromogens that work with each of the enzymes. 
> 
> If the antibodies are from the same species I see no way to distinguish 
> both. Only if one is conjugated with biotin and the other with digoxigenin, 
> then you could proceed with secondaries against biotin and digoxigenin. 
> etc.. 
> 
> Gudrun 
> 
> -Ursprüngliche Nachricht- 
> Von: Judi Ford via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
> Gesendet: Samstag, 20. Februar 2016 01:54 
> An: histonet@lists.utsouthwestern.edu 
> Betreff: [Histonet] Double stain IHC question 
> 
> Hi everyone, 
> I have a question in chromogenic double staining. Here is the situation. 
>                 Tissue = human, frozen 
>                 Antibody = same protein (A) 
> 
> 1.       Commercial antibody of A 
> 
> 2.       Homegrown antibody of A, human, biotinylated 
> 
> Question: can you stain both versions of this antibody on the same tissue, 
> same slide? Goal is to see where each stains in the tissue and if they 
> co-localize. If they do co-localize then how do you distinguish between 
> that 
> and where they stain individually? Would you use different chromogens and 
> hope that where they come together it turns a different color? 
> I am really interested if this can work. Thanks in advance for any replies. 
> Judi 
> South San Francisco, CA 
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-- 
Caroline Miller (mills) 
Director of Histology 
3Scan.com 
415 2187297 
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Re: [Histonet] Histonet Digest, Vol 147, Issue 21"............Double stain IHC question

[Dorothy Hu via Histonet]

I wonder if streptavidin Alexa (495 or 488) can be use to bind to biotinlyted 
primary. So no worry about species issue? May need to do biotin blocking first 
to get ride of endogenic biotin I guess. 

Dorothy Hu


Sent from my iPad

> On Feb 20, 2016, at 1:00 PM, histonet-requ...@lists.utsouthwestern.edu wrote:
> 
> Double stain IHC question

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[Histonet] Positive Streptococcus pneumoniae IHC controls

[Kristina Wyatt via Histonet]

Does anyone know of a commercial positive control tissue for S. pneumoniae? We 
have  a pig study and need an antibody along with a known positive control to 
run optimization tests. So far, I have a reference for the antibody used in 
mouse models, but can't track down positive tissue for a control.


Thanks,


Kristina Wyatt, M.A.
Supervisor: Histopathology/Immunohistochemistry
L216 Mosier Hall
Kansas State Veterinary Diagnostic Laboratory
785-532-4464
kdwy...@vet.k-state.edu
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[Histonet] question - Allergy to histological solvents?

[daniel blackburn via Histonet]

Hello

After decades of using standard organic solvents for paraffin- section 
histology, I find that I've become highly allergic to the fumes.  These include 
commercial preps like Citrosolv and Hemo-De, as well as xylene and toluene, 
which cause (in me) dizziness and a pounding headache.  This problem has made 
staining and coverslipping very difficult, even with  hood.   Can anyone 
recommend alternative solutions for (a) dewaxing prior to staining and (b) 
coverslipping?  thanks very much!

Daniel Blackburn
Biology, Trinity College (Hartford CT)

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Re: [Histonet] Double stain IHC question

[Caroline Miller via Histonet]

+ to Gudrun's comments.

My addition is that fluorescence labels may work for this application.
Again, depending on the species of the antibodies. The other advantage of
the fluorescence is that you would be able to truly see the colocalization
(yellow for instance if you used red and green fluorescent antibodies).
Rather than the muddiness I have often got when trying two antibodies in
bright field chromagenic stains.

Remember though autofluorescence is greatly increased with paraffin
tissues, that may put another issue on your plate.

Good luck!

yours,
mills

On Sat, Feb 20, 2016 at 3:47 AM, Gudrun Lang via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> In my opinion, this would only be possible, if the commercial and the
> homegrown antibody are from different species. For example one from mouse
> and one from rabbit.
> Then you can proceed with different secondaries (goat anti mouse conjugated
> with peroxidase, goat anti rabbit conjugated with alkaline phosphatase).
> Then chromogens that work with each of the enzymes.
>
> If the antibodies are from the same species I see no way to distinguish
> both. Only if one is conjugated with biotin and the other with digoxigenin,
> then you could proceed with secondaries against biotin and digoxigenin.
> etc..
>
> Gudrun
>
> -Ursprüngliche Nachricht-
> Von: Judi Ford via Histonet [mailto:histonet@lists.utsouthwestern.edu]
> Gesendet: Samstag, 20. Februar 2016 01:54
> An: histonet@lists.utsouthwestern.edu
> Betreff: [Histonet] Double stain IHC question
>
> Hi everyone,
> I have a question in chromogenic double staining. Here is the situation.
> Tissue = human, frozen
> Antibody = same protein (A)
>
> 1.   Commercial antibody of A
>
> 2.   Homegrown antibody of A, human, biotinylated
>
> Question: can you stain both versions of this antibody on the same tissue,
> same slide? Goal is to see where each stains in the tissue and if they
> co-localize. If they do co-localize then how do you distinguish between
> that
> and where they stain individually? Would you use different chromogens and
> hope that where they come together it turns a different color?
> I am really interested if this can work. Thanks in advance for any replies.
> Judi
> South San Francisco, CA
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>
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-- 
Caroline Miller (mills)
Director of Histology
3Scan.com
415 2187297
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Re: [Histonet] Double stain IHC question

[Gudrun Lang via Histonet]

In my opinion, this would only be possible, if the commercial and the
homegrown antibody are from different species. For example one from mouse
and one from rabbit.
Then you can proceed with different secondaries (goat anti mouse conjugated
with peroxidase, goat anti rabbit conjugated with alkaline phosphatase).
Then chromogens that work with each of the enzymes. 

If the antibodies are from the same species I see no way to distinguish
both. Only if one is conjugated with biotin and the other with digoxigenin,
then you could proceed with secondaries against biotin and digoxigenin.
etc..

Gudrun

-Ursprüngliche Nachricht-
Von: Judi Ford via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Gesendet: Samstag, 20. Februar 2016 01:54
An: histonet@lists.utsouthwestern.edu
Betreff: [Histonet] Double stain IHC question

Hi everyone,
I have a question in chromogenic double staining. Here is the situation.
Tissue = human, frozen
Antibody = same protein (A)

1.   Commercial antibody of A

2.   Homegrown antibody of A, human, biotinylated

Question: can you stain both versions of this antibody on the same tissue,
same slide? Goal is to see where each stains in the tissue and if they
co-localize. If they do co-localize then how do you distinguish between that
and where they stain individually? Would you use different chromogens and
hope that where they come together it turns a different color?
I am really interested if this can work. Thanks in advance for any replies.
Judi
South San Francisco, CA
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