The rats (20 months-old) were deeply anesthetized with sodium pentobarbital and transcardially perfused with 4% paraformaldehyde. The brains were included in paraphin and cut in a microtome at 20 microns.
We and many others have had great success using an Iba-1 antibody from Waco. It is specific for both rats and mice. For the rat the antibody seems give very good labelling at a concentration of 1:10,000 or greater and for mice its 1:1000-1:5000. See any of our groups papers on microglia for a basic outline of the method, and if you would like specifics (what was buffers, wash times, incubation times etc) just email me and I will send you our detailed protocol.
What is the best dilution for GR (polyclonal rabbit antibody, M-20, Santa Cruz Biotechnology)? The datasheet recomend a dilution between 1:50 and 1:500, but I do not know what is the most recommended.
Perhaps I am misunderstanding from past answers, but I believe that you asked for a protocol for paraffin-embedded tissue... I embed my tissue in PEG, which is very similar to paraffin and includes similar antigen retrieval steps. I also use DAB as my chromagen, but if you are going to use something fluorescent then remove the unnecessary steps.
The protocol is:
Day 1:
1. Wash in PBS
2. Immerse in 37 degree C PBS (5 mins, but flexible)
3. .1N HCl with 1.2g pepsin for exactly 3 mins at 37 degrees C. (This step will make of break my protocol) This is the only antigen-retrieval step that I use.
4. Wash in room temperature PBS (all remaining steps are done at room temp unless specified)
5. Block endogenous peroxidase with .3% methanol, .3%H2O2 and PBS
6. Wash in PBS
7. Apply primary antibody (Wako) 1:100 with .3% Triton X, 2% normal serum and PBS. Vortex
8. I horizontally apply the antibody (400uL per slide) covered with slips, in a hydration chamber and leave them overnight in the fridge.
Day 2:
1. Wash off the primary with PBS
2. Block endogenous avidin with avidin block for 30 mins
3. Rinse with PBS
4. Block endogenous biotin with biotin block for 30 mins.
5. Rinse with PBS
7. Apply secondary antibody (1:200) for 2 hours in a hydration chamber with .3% triton X, 2% normal serum and PBS.
8. Wash in PBS
9. Apply ABC VectorKit, 1 part A, 1 part B into buffer of choice. (Must have been left at room temp for 30 minutes prior). Let incubate for 30 mins.
10. Wash in PBS
11. Dehydrate and coverslip in a series of alcohols followed by xylenes. I use 70uL of Krystalon to coverslip my slides.
The critical steps for you (using paraffin at 20u) will be to get the antigen-retrieval steps correct, as well as the permeation of the tissue by the Triton X. Otherwise the protocols from people who use fresh/fresh-frozen will be useful as well. Just tweek theirs with whatever retrieval steps work for you.
Troubleshooting is so much fun! (:
Good luck!
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