Paraformaldehyde Perfusion and Immunohistochemical Analysis of P-gp and HIF-1α in Frozen Heart Sections

After deep anesthesia with ketamine/xylazine (90/10 mg/kg), animals were sacrificed by intracardiac perfusion with 4% paraformaldehyde in phosphate buffer as previously described [29 (link)]. Frozen hearts were cut into 14-micron sections and mounted on slides. Subsequently, the sections were dehydrated to perform the inhibition of endogenous peroxidase with H₂O₂ (0.5% v/v in methanol) for 30 min at room temperature; then rehydrated and permeabilized with 1% Triton X-100 in buffer phosphate saline (PBS) for 30 min. Nonspecific sites were blocked with 3% equine normal serum in PBS for 30 min, and incubation with anti-P-gp (dilution: 1/500; EPR10364-57 Abcam Inc., Cambridge, MA, USA) was performed for 48 h at 4 °C. Subsequently, incubation with a biotinylated secondary antibody (dilution 1/800; B7389 Sigma, St. Louis, MO, USA) was performed for 3 h at room temperature. Immunoreactivity was revealed with diaminobenzidine/nickel until coloration was observed and mounted with DPX (Distrene-Plasticizer- Xylene) mounting medium. Slides were also treated with monoclonal antibody anti-HIF-1α (dilution: 1/500 NB-100-131 NovusBio, Littleton, CO, USA) and revealed by immunofluorescence using a labelled Alexa488 donkey anti-mouse (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).