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Immunohistochemistry Question

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Mrbojangles

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I have a question, can double immunostaining be done using chromogens? For instance if you want to stain the same cell with two antibodies. I've only heard it being down using fluorescent antibodies.
 

djmd

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I have a question, can double immunostaining be done using chromogens? For instance if you want to stain the same cell with two antibodies. I've only heard it being down using fluorescent antibodies.


Yes... You can used both AAC and DAB with different antibodies...
I have seen it done for prostate with AMACR & HWM-CK.

(this is a Breast case for ck5 & p63)
CK5___p63_with_CK8_18_Double_Stain_Cocktail.jpg
 

Mrbojangles

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But can you actually stain the same cell with two antibodies? For a mix of brown and red. Sort of like flow cytometry. I'm wondering if I can stain plasma cells with CD138 and CD56...
 

djmd

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But can you actually stain the same cell with two antibodies? For a mix of brown and red. Sort of like flow cytometry. I'm wondering if I can stain plasma cells with CD138 and CD56...

That seems like that would be hard to read...

I think that has to be two different slides... I don't know how well you could read two overlapping cytoplasmic stains..
(if it was a nuclear and cytoplasmic (or membranous and cytoplasmic))
 

Mrbojangles

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That seems like that would be hard to read...

I think that has to be two different slides... I don't know how well you could read two overlapping cytoplasmic stains..
(if it was a nuclear and cytoplasmic (or membranous and cytoplasmic))

Yeah I was wondering if it is possible too. I know it can work for fluorescence tagged antibodies (red + green = yellow) but haven't seen it yet for chromogen tagged antibodies.
 

LADoc00

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But can you actually stain the same cell with two antibodies? For a mix of brown and red. Sort of like flow cytometry. I'm wondering if I can stain plasma cells with CD138 and CD56...

yeah...its done like everyday. Do some research on it.
If they stain different cellular compartments ie-nuclear vs. membranous, they are easily read by anyone not completely brain dead.
 

pathstudent

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yeah...its done like everyday. Do some research on it.
If they stain different cellular compartments ie-nuclear vs. membranous, they are easily read by anyone not completely brain dead.

Do you get to bill for 2 88342s if they are on the same slide?
 

Circumflex

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It can be done, but visual interpretation can be difficult, depending on the pattern of staining. There are imaging systems available that will acquire a specific wavelength and subtract all others, so you can get an image of each chromagen separately, then combine to see which cells are double-positive.
 

PathJet

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Yeah I was wondering if it is possible too. I know it can work for fluorescence tagged antibodies (red + green = yellow) but haven't seen it yet for chromogen tagged antibodies.


pubmed ?:idea:

try PIN cocktail
 

Mrbojangles

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yeah...its done like everyday. Do some research on it.
If they stain different cellular compartments ie-nuclear vs. membranous, they are easily read by anyone not completely brain dead.

I just haven't come across it. And no I'm not talking about cocktail stains like PIN4 which stains different cell types.
 

Mrbojangles

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It can be done, but visual interpretation can be difficult, depending on the pattern of staining. There are imaging systems available that will acquire a specific wavelength and subtract all others, so you can get an image of each chromagen separately, then combine to see which cells are double-positive.

The antigens I'm primarily interested in (in addition to CD138) are cytoplasmic. I'll have to check if the Immage Analysis in my dept can do that.
 

docbiohazard

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So fluorescence isn't an option? Because I imagine it'd look really nice with some multicolor confocal imaging...

BH
 
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