Immunohistochemistry with SC48 Antibody

Protocol Overview

1) Rats received a transcardial perfusion of 50ml of cold saline (0.9%) to remove excess blood from the major arteries. 2) Rat brains were transcardially fixed in vivo by a transcardial infusion of 4% paraformaldehyde in Phoshate buffered solution. 3) Brains were isolated and postfixed in 4% paraformaldehyde solution overnight at 4oC. 4) Brains were washed with PBS buffer and dropped and incubated in a 30% sucrose in PBS solution for 5 days for cryoprotection. 5) Brains were dissected sagitally 2mm lateral to the midline, and frozen in OCT compound. 6) The frozen blocks were sectioned with a cryostat at 40um sections. 7) The sections were collected in PBS. 8) Sections treated with 0.1% H2O2 for 30minutes 9) Sections washed 3X3' in PBS. 10) Section blocked with 5%NGS in PBS containing 0.3%TritonX-100 for 2hours on a rocking platform at room temperature. 11) Sections washed 3x3' in PBS12)Applied Primary antibody diluted at 1:600 in PBS containing 1%NGS and incubate on rocking platform overnight at 4oC. 13) Washed sections 3X3' in PBS 14) Used Vectastain ABC kit PK-4000 as per kit instructions to conjugate primary with biotinylated secondary to heighten signal. 15) Washed sections with PBS 3X3'. 16) DAB with 0.015% H2O2 was used to develop staining.