[Histonet] Mouse FFPE IHC double stain caspase-3 and Ki67

[Melanie Smith]

Hi everyone,



My lab is trying to do caspase-3 and Ki67 double IHC stain on mouse ffpe
knee joint sections, and we're having issues optimizing the caspase-3
staining. We originally tried to do fluorescent labels but had too much
autofluorescence even after trying several methods of knocking it down. We
decided to move on to traditional IHC and purchased a double stain IHC kit
from abcam R&Rt on human/mouse tissue (DAB & AP/Red) (
http://www.abcam.com/doublestain-ihc-kit-ramprt-on-humanmouse-tissue-dab-amp-apred-ab183283.html).
We started by tying to optimize both antigens independently first and had
success with the Ki-67 staining (primary rat anti-ki67, eBioscience) with
the abcam anti-rat secondary HRP polymer and DAB from the kit. However, we
still haven’t had any luck with the caspase-3 (primary rabbit anti-caspase
3, cell signaling & abcam anti-rabbit secondary AP polymer and permanent
red). We also tried the caspase-3 staining in cell culture after treating
cells with an apoptosis inducer, and only saw extremely weak staining. In
the FFPE sections, we have used overnight antigen retrieval in citrate
buffer ph 6, at 60 C. In both the cell and FFPE sections we do an overnight
primary antibody incubation at 4C. We haven’t seen any convincing staining
in our FFPE sections (there is some dark pink staining but only in one or
two bone marrow cells per section and it seems pretty diffuse) and also
have noticed a yellow/brown precipitate forming and only showing up in the
bone marrow, it also shows up in our negative antibody control sections. We
also tried several different chromogen development times ranging from 10
min to an hour, and didn’t really see a difference. All sections are of
course deparaffinized in xylenes and rehydrated in ethanols before
starting. All washes are done with TBS-T and sections are permeabilized in
0.1% Triton X100 and blocked in 1%BSA in TBST before primary incubation.
All sections are blocked with BLOXALL Endogenous Peroxidase and Alkaline
Phosphatase Blocking Solution from Vector after primary incubation, but
before secondary incubation.


If anyone has any insight into caspase-3 antibodies or doing IHC double
staining that would be great!


Many thanks,

Melanie

-- 
Melanie Smith, MS
Research Technician II
Department of Biomedical Engineering
University of Delaware
melsmith <@t> udel.edu