Caroline,
Overnight at 4 degrees may not be long enough for equilibration of the
antibody binding reaction deep in the section. Try room temp or longer
time if cold is important. The early users of IHC often used 4 days at
4 deg C, based on antibody binding kinetics data. I get full-thickness
labeling assessed by confocal microscopy with overnight incubations
(both primary and secondary) at room temp in 50 um rodent brain
sections. Your secondary incubation may be too short for deep
labeling--easy to test. Alternatively, the primary may be depleted (all
bound to tissue epitopes) before penetrating the section interior.
Increasing the volume or concentration of primary would help. Some
24-well plates (culture-treated) will absorb significant (and variable)
amounts of antibody, and little critters can gobble it up too. If you're
not using peroxidase reporters you may want to leave azide or another
antimicrobial in the incubation solutions. They don't alter
fluorescence, though you'll want to convince yourself of this
empirically.
----
Message: 12 Date: Tue, 19 Nov 2013 17:43:13 +0000 From: "Bass,
Caroline" <ceb...@buffalo.edu> Subject: [Histonet] problem with double
IF of brain section To: Histonet <histonet@lists.utsouthwestern.edu>
Message-ID: <3ccee4fb-40b5-49a2-8456-72d71f893...@buffalo.edu>
Content-Type: text/plain; charset="Windows-1252" Hello everyone, I’m
trying a double stain for GFP and tyrosine hydroxylase in floating brain
sections. Rat, perfused with formalin. I’m having a big problem, in
that it’s clear that the top side of the sections are well stained, as
are the bottom, but everything in between is not. These sections are 50
microns. Any suggestions? I assume it’s a penetration problem. But
I’m not very comfortable with IF in general. Here’s what my tech
does: Placed sections each in a well of a 24 well dish and rinsed in PBS
(to remove the Na azide), 1 X 5 min. Rinsed in PBS-0.5% Triton X-100, 3
X 10 min. Incubated tissue in PBST (0.25% Triton X) + 5% NGS, 1 X 60 to
120 min. Incubated sections in 1ary antibody in PBS-T(0.25%) overnight
@40C (covered to minimized evaporation). Both primaries together.
Tyrosine Hydroxylase 1: 4,000 ImmunoStar #22941 (mouse) a- GFP 1:2,000
Invitrogen 6455 (rabbit) Next day, rinsed tissue w/ PBS-T, 3 x 10 min.
Incubated w/ 2ary ab : Alexa 555 donkey anti mouse 1:4,000, Alexa 488
goat anti rabbit, 1:2,000, 1 x 2hs @RT. 2ary ab diluted in PBS. Rinsed
w/ PBS, 3 x10 min. Mounted on slides, coverslipped using ProLOng Gold w/
DAPI. Cured in dark place @ RT overnight. Thanks, Caroline
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