Example of Immunohistochemistry protocol
Perfusion protocol for Adult male Sprague Dawley (weight around 0.5 kg) :
1-The animals can be deeply anaesthetized for example with urethane (0.5-1.5g/kg, intraperitoneal).
2-Heparinized, and perfused via the ascending aorta with 100 ml of cold physiologic saline (0.9% NaCl) and with the
following fixative solution:
a) 300 ml of cold 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate-buffer (PB), pH 7.2, (two
b) 600 ml of cold 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate-buffer (PB), pH 7.2, (ten
c) Dissect out the brains and place in a solution of 4% paraformaldehyde in 0.1M PB, pH 7.2, at 4ºC for twelve to
d) Before the brains will be cut on a freezing microtome, we must include the brain in growing concentrations of
sucrose (a first bain of 5% of sucrose in PBS until the brains sank), after that we will repeat the same process in a
solution with a higher level of sucrose (10%), 20%, 25% and finally 30%.
Around 50 μm-thick serial sections will be obtained, kept at 4o C in PBS (0.1 M, pH 7.2) and processed for immunostaining.
Example of immunohistochemical protocol
1-In order to avoid possible interference with endogenous peroxidase, free-floating sections will be treated with distilled
water containing NH3 (20%), H2O2 (30%) and NaOH (1%) for 20 min (other method is using a solution with 33% of H2O2
and 66% of methanol).
2-Then, wash the sections for 20 min in 0.15 M phosphate-buffered saline (PBS) (pH 7.2)
3-Pre-incubate for 30 min in PBS containing 10% of normal horse serum and 0.3% of Triton X-100 (mixed solution).
4-Incubate at room temperature (1h 30min) and overnight at 4ºC in the same mixed solution containing anti-cojugated spermine antibodies (diluted 1:500 to 1:1000; as recommended dilution).
5-Then, the sections will be wash in PBS (30 min).
6-After that we will incubate for 60 min at room temperature with biotinylated anti-rabbit immunogammaglobulin (Vector) diluted 1/200 in PBS.
7-Wash during 30 min with PBS.
8-Sections will be incubated for 1 h with a 1:100 diluted avidin-biotin-peroxidase complex (Vectastain).
9-After that we will wash the sections in PBS (30 min)
10-Wash with Tris-HCl buffer (pH 7.6)(10 min).
11-The tissue-bound peroxidase will be developed with H2O2 using 3, 3’ diaminobenzidine as chromogen.
12-Finally the sections will be rinsed with PBS and coverslipped with PBS/Glycerol (1/1).