IHC Related Problem

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Mon Dec 17 10:23:18 EST 2007


Hello all,
We are trying to do IHC on free floating 30 microns rat brain sections.
Primary is antiGFAP (Mouse Monoclonal-1:1000) and secondary is antiMouse
(Horse: 1:250), by ABC-DAB method.
But we are getting reproducible nonspecific staining in SPECIFIC regions of
the rat brain

Our IHC method is
1. TBS / PBS Washing
2. Quenching for 10 min
3. TBS/ PBS washing
4. Blocking with 3%-5% Normal Horse Serum (also tried BSA)
5. Washing
6. Primary
7. Washing
8. Secondary
9. Washing
10. ABC
11. Washing
12. DAB

We did a negative control by skipping the primary..but same results..the
cells seem to be of pyramidal / immature neuron morphology.
We also skipped both primary and secondary, going ahead with ABC-DAB but no
staining (rules out endogenous biotin).

We got convinced that only secondary can give nonspecific staining..we tried
following methods-(Quenching-Blocking-Secondary)
                 1. increasing dilutions: from 1:250 to 1:1000 and then
ABC-DAB..still we are getting the same nonspecific staining..
                 2. Texas Red conjugated antiMouse (Horse) as secondary and
got the similar pattern (no ABC method)
                 3. used antiRabbit(Goat) without primary..no staining
(looks like problem is specific to antiMouse)
                 4. Reagents were changed, new vials (lots) were used,
crucibles were changed..6-7 different brain sections were used..same result
                 5. This post-secondary antibody stain looks predominantly
cytoplasmic, also present in axons
                 6. regions where staining is seen : somato sensory cortex,
hippocampus, inferior temporal lobe, SVZ etc
                 7. Increasing blocking serum (Horse/Goat): 3 to 5%--same
results
                 8. We have also tried NovaRed--same results.

So, this antimouse antibody is binding to some proteins expressed in normal
rat brain..??

in dire need of help.

regards
Deepti and Sudheendra
NBRC, Gurgaon, India


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