Re: [Histonet] Bright field and fluorescence staining
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:
>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
>Thanks a lot!
>Unless expressly stated otherwise, this message is confidential and may be
>privileged. It is intended for the addressee(s) only. Access to this
>E-mail by anyone else is unauthorized. If you are not an addressee, any
>disclosure or copying of the contents of this E-mail or any action taken
>(or not taken) in reliance on it is unauthorized and may be unlawful. If
>you are not an addressee, please inform the sender immediately.
>Histonet mailing list
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-4303 (FAX)
Histonet mailing list
<< Previous Message | Next Message >>