immunofluorescent IHC

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From:Gayle Callis <>
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For cell markers, we work with the same concentration of antibody, generally


1.Blocking can be the same, PBS with normal serum matching the host species
of secondary, 1-5% for 15 - 30 min, depends on the antibody. Tween is
generally not used

2.The primary can be a directly conjugated fluorchrome, used for FACS work

3. OR a purified antibody, ascites, or supernate WITH Biotinylated
secondary followed by Strepavidin flurochrome, we like Alexa fluorochromes
from Molecular Probes, you will need to determine the optimal dilution for
your fluorochromes (panel dilutions) spec sheets or tech services give the
general ranges to start with ug/ml

4. a directly conjugated secondary antibody with fluorochrome,
FITC/TRITC/Alexa's, you will need to determine optimal dilution for these,
Jackson has excellent ones, F(AB')2 are preferred here with mouse or rat

PBS works great, we often put 0.2% BSA or 0.1-0.2% goat serum in the
buffer, and also add 0.01M sodium azide to fluorochrome buffers.  Buffers
for the fluorochromes vary, Alexa is recommended with PURE buffer, nothing

The final rinse is 5 to 6 changes pure buffer, coverslip with a mounting
media appropriate for maintaining fluorescence and less quenching.  

Some rules

IFA double
1.  first antibody combination  primary, then fluorochrome conjugated

2.  Direct conjugated primary 

This helps override "bleedover" on different filters during viewing.

I don't know if this helps, but we actually do less blocking, more rinsing
with IFA, and all incubations the same as IP or Alk Phos, in the DARK!
Covered with foil, whatever works. 

Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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