gcallis <@t> montana.edu
Wed Nov 8 10:27:36 CST 2006
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.
At 07:42 AM 11/7/2006, you wrote:
>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.
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