Re: [Histonet] Bright field and fluorescence staining

From:Gayle Callis

I suggest using an alkaline phosphatase immunostaining method and then 
DAKO's permanent red. Counterstain with JUST hematoxylin.  Chris van der 
Loos taught me to underdevelop the permanent red so have very delicate 
fluorescence but you need to control the development of this chromogen (a 
very sensitive one for AP!) with a microscope.  If you let it go to a 
darker red endpoint, you still have fluorescence but more diffuse, even 
though this works well for bright field.  You can adjust for how to view in 
both modes.   Vector red also fluorescences, and it too needs 
underdevelopment.  We find the DAKO a bit more sensitive than the Vector 
red, but if the antigen/antibody complex is plentiful and strong, you could 
try either.  Be sure to use Tween 20 in the buffer with Vector red, it 
cleans up things and makes the VR sharper.

We use it with polymer kits to have biotin free IHC, but you can use PR 
with any AP IHC method.  Poymer kits are extremely sensitive, so be careful 
about antibody dilutions, etc - look at kit instructions.  We use the 
ImmunoVision aka Powervision now sold by VisionBiosystems and also trying 
the new Biocare polymer system for rodents.

The contrast with hematoxylin is excellent and without interference with 
this stain but eosin can't be used since it fluoresces and will probably 
interfer with the PR fluorescence.

At 07:50 AM 8/22/2006, you wrote:
>Hallo everyone,
>I'm posting this for a colleague of mine.
>Can someone help her with the staining question???
>Do you know any classical bright field staining, such as H&E or Geimsa
>(ideal to distinguish blood cells) but would not interfere with
>fluorescence? Ideally, we need to do the immunostaining, then bright
>field stain, and be able to look at both fluorescence signal and bright
>field signal.
>Thanks a lot!
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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