Rabbit anti neun

Brain sections were dewaxed with xylene and dehydrated by ethanol at graded concentrations followed by distilled water. Sections were then incubated for 10 min in 3% hydrogen peroxide to block endogenous peroxidase activity. High‐temperature (boiled buffer) antigen retrieval was performed in a 0.01 m citrate buffer at a pH of 6.0 for 20 min. Brain sections were then incubated overnight at 4 °C with mouse anti‐ICAM1 (CD54), immunoglobulin G (IgG) (diluted 1 : 200, Thermo, Carlsbad, CA, USA), rabbit anti‐NeuN (diluted 1 : 500, Proteintech, Rosemont, IL, USA), or mouse anti‐BDNF IgG (diluted 1 : 200, Abcam, Cambridge, UK) in PBS containing 0.3% (v/v) Triton X‐100, followed by incubation in 37 °C EnVision (Zymed, South San Francisco, CA, USA) solution for 30 min. Finally, sections were incubated with peroxidase substrate diaminobenzidine until a desired staining intensity developed, followed by slight counterstaining with hematoxylin, dehydration, and cover‐slipping with permount. Between incubations, the tissue was washed with PBS three times for 10 min each. Photographing was performed using a microscope (Olympus BX43F, Tokyo, Japan) and ProgRes CapturePro software (Jenoptik Laser GmbH, Jena, Germany).