[Histonet] CD 102 (ICAM2)

[Bascaramurty, Saro]

Hi,

I would like to get some help on labelling CD102 by an
immunohistochemical method on mouse heart after it has gone through
Langendorff perfusion with ice-cold Krebs-Henseleit buffer during an
experiment. We have purchased BD Pharmingen's 'Purified Rat-Anti-Mouse
CD102' and 'Anti-Rat Ig HRP detection kit'. Will be using normal goat
serum for blocking the non-specific binding.These reagents have been
tested in frozen sections only by BD. Since I have not done that many
immunohistochemical assays, I would like to get answers to the following
from the experts:

1. Has anybody done this on paraffin embedded tissue sections using the
same reagents?

2. If using frozen, how do you prepare the tissue? Do you fix the tissue
before freezing or soon after it has been sectioned? Do you use 4%
paraformaldehyde or of a lesser concentration? Do you cryo-protect using
sucrose in PBS? If so, sucrose is used at what percentage?

3. What's the thickness range of the tissue section recommended for the
frozen tissue?

4. What kind of dilutions I need to include for the primary antibody and
for the secondary antibody for my initial trial run during optimization?

5. What are the incubation times and temperature for the primary and
secondary antibodies?

6. Which tissue type would be the best positive control to use in the
assay?

Any kind of help with this protocol would be greatly appreciated!

Thank you so much! 

Saro Bascaramurty

Technical Officer
Institute for Biodiagnostics
National Research Council
435 Ellice Avenue,
Winnipeg, Manitoba. R3B 1Y6

Tel: 204-984-7166
Fax:204-984-6978
email:saro.bascaramurty <@t> nrc-cnrc.gc.ca