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

[James Watson]

Sorry forgot to add loner HIER damaged the Caspase 3 antigen.

James Watson HT  ASCP
GNF  Genomics Institute of the Novartis Research Foundation
Scientific Technical Leader II, Histology
Tel    858-332-4647
Fax   858-812-1915
jwatson <@t> gnf.org


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of James Watson
Sent: Wednesday, October 08, 2014 8:52 AM
To: Melanie Smith; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Mouse FFPE IHC double stain caspase-3 and Ki67

Melanie,

We do this stain,  we run Ventana stainers (no advertisement for Ventana intended) .  We had to do this as a sequential double stain since the Caspase 3 needed polymer detection where the Ki67 only needs ABC detection.  Caspase 3 was done first.  Some other differences:  Caspase 3 needed FC receptor block and permeablization.  Ki67 needed longer HIER time than Caspase 3 so we did a second short HIER after the Caspase 3 was labeled with DAB.  

Jamie

James Watson HT  ASCP
GNF  Genomics Institute of the Novartis Research Foundation Scientific Technical Leader II, Histology Tel    858-332-4647 Fax   858-812-1915 jwatson <@t> gnf.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Melanie Smith
Sent: Wednesday, October 08, 2014 8:38 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Mouse FFPE IHC double stain caspase-3 and Ki67

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
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