Ab53088

Rat brains were postfixed in 4% paraformaldehyde and cryoprotected in 30% sucrose. The rat brains were sectioned coronally at 30μm using a freezing microtome. Free-floating sections were rinsed with phosphate buffered saline (PBS) with 0.3% TritonX-100 and blocked with 10% normal goat serum in PBS. After blocking brain sections were incubated with mGlu₄ (1:500, ab53088, Abcam) and mGlu₅ (1:1000, ab76316, Abcam) antibody overnight. The tissue was then rinsed in PBS with 0.3% TritonX-100 and incubated for 1 hour in biotinylated secondary antibody (1:200, Vector Labs, Burlingame, CA, USA). After secondary antibody sections were incubated for 1 hour in ABC (Vector Labs) solution at RT. Following incubation, sections were rinsed with PBS and were visualized by incubating in 3,3′-diaminobenzidine (DAB) substrate kit Vector Labs).
To quantify mGlu₄ sections from the lesion and non-lesion side of the hippocampus were counted using the Optical Fractionator probe in the StereoInvestigator software (MBF Bioscience, Williston, VT) for each group (n=3). The region of interest was delineated at 4× magnification. A counting grid of 250×250 μm was placed over the hippocampus. Using 100×100 μm counting frame mGlu₄ immunoreactive cells were counted in randomly-placed sampling sites with 40× magnification.