Ab3573

Anesthetized 10-day-old C57Bl/6 mice were perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde. Brains were collected and cut into 40-μm-thick frozen sections. The sections were blocked with 10% normal serum and incubated with rabbit anti-cytochrome P450 4A (CYP4A) antibody (1:300; ab3573, Abcam, Cambridge, MA) or mouse anti-NeuN (1:500, MAB377, MilliporeSigma) and mouse anti-VR1 (1:300, sc-398417, Santa Cruz Biotechnology, Dallas, TX) overnight at 4 °C, followed by 1-h incubation with anti-IgG antibodies conjugated with Alexa Fluor 488 or 594 (1:1000, ThermoFisher).
Paraformaldehyde-fixed cells (on glass coverslips) were washed with PBS, permeabilized with 0.1% Triton X-100 (MilliporeSigma), blocked for 30 min with 10% normal goat serum in PBS, and incubated with rabbit anti-CYP4A antibody and mouse anti-VR1 antibody overnight at 4 °C, followed by 1-h incubation with anti-rabbit or anti-mouse IgG antibodies conjugated with Alexa Fluor 488 or 594.

Anesthetized naïve piglets were perfused transcardially with ice-cold phosphate-buffered saline (PBS) and 4% paraformaldehyde. The right forebrain was cryoprotected in 20% glycerol-PBS for 24 hours, frozen, and cut into serial 60-μm sections. Immunohistochemistry was completed on free-floating sections to detect CYP 4A in putamen, primary sensorimotor cortex, hippocampus, and ventral posterolateral (VPL) thalamus. Endogenous peroxidase was quenched with 30-minute incubation of 1% H₂O₂ in methanol. Sections were blocked in normal serum and incubated with rabbit anti-CYP 4A (1:250; Abcam ab3573, Cambridge,UK) overnight at 4°C. After rinsing in PBS, sections were incubated with biotinylated anti-rabbit IgG (1:200, Vector Laboratories, Burlingame, CA, USA) and VECTASTAIN Elite ABC reagent (Vector). Immunostaining was developed with diaminobenzidine as a chromogen (Vector). Negative controls were treated without primary antibodies and showed no positive signals.

The animal protocol was approved by the Animal Care and Use Committee of the Johns Hopkins University. In brief, anesthetized C57Bl/6 mice (7 days old, Charles River, Frederick, MD) were perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde. Brains were collected and cut into 40-μm-thick frozen sections. The sections were blocked with 10% normal goat serum and incubated with rabbit anti-cytochrome P450 4A (CYP4A) antibody (1:300; ab3573, Abcam, Cambridge, MA) and mouse anti-NeuN (1:500, MAB377, EMD Millipore, Billerica, MA) overnight at 4°C, followed by 1 h incubation with anti-IgG antibodies conjugated with Alexa Fluor 488 or 594 (1:1000, Life Technologies, Grand Island, NY).