Re: [Histonet] F4/80

From:Gayle Callis


You may need to retiter the primary for immunofluorescence (IFA) staining, 
generally we find an increase in primary antibody concentration.

You will have less formalin induced autofluorescence if you use cryostat 
sections which also eliminates retrieval. On a fresh tissue cryostat 
section,  fixation with either 75% acetone/25% absolute ethanol for 5 min 
at RT on overnight dried frozen sections then go directly to pure buffer 3 
changes.  OR 4C acetone fixation for 10 minutes but air dry after pure 
acetone fixation. Both will work nicely.

If you do FFPE tissues, retrieval and have problems with aldehyde induced 
autofluorescence, just us a red fluorophore which allows the 
autofluorescence to become a "counterfluorescence" .

You can use Goat antiRat conjugated to FITC, RRX (rhodamine extra) or even 
a donkey anti Rat conjugated to a fluorophore of choice from Jackson.  We 
prefer F(ab')2 frag of IgG antibodies, adsorbed to mouse to prevent binding 
to fc receptors on mouse tissues to give cleaner staining.  Another source 
of a secondary is Molecular Probes, and find one adsorbed to mouse 
conjugated to Alexa 488 (FITC substitute) or Alexa 594 (Texas Red 
substitute).  The reason for 594 is if you ever do double IFA with FITC, 
you have better separation these two fluorophores.

  The Alexa dyes are very bright and do not photobleach excessively.  Be 
sure to use an antifade mounting media, Prolong Gold antifade reagent, 
ready to use hard set from Molecular Probes is excellent and if you want 
DNA stained, you can buy this with DAPI already in the mounting media.

Good luck

At 07:42 AM 11/7/2006, you wrote:
>Hi Histonetters
>I have been able to stain some mouse liver for Kuppfer cells using
>Serotec's F4/80 and copious amounts of help from their tech support,
>however I would like to use some immunofluorescence instead of the DAB.
>At present I am using a Goat anti Rat IgG( Mouse adsorbed) :HRP
>secondary.  Does anyone know of another secondary I could use with a
>fluorescent marker.  I am using FFPE or cryostat sections.
>Histonet mailing list

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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