Avidin biotin horseradish peroxidase complex abc

BBB integrity was assessed by IgG immunostaining with minor modifications [23 (link)] and Texas Red^® (Invitrogen, Carlsbad, CA, USA) dextran perfusion [24 (link)]. Free-floating sections were incubated with biotinylated horse anti-mouse IgG (BA-2000, 1:500, Vector Laboratories) at 4°C overnight following blocking in 10% normal horse serum and 1% BSA in PBS, pH 7.4. After rinsing in 0.3% H₂O₂ for 30 minutes to quench endogenous peroxidase activity, sections were incubated with avidin-biotin horseradish peroxidase complex (ABC, Vector Laboratories) for 30 minutes, then visualized with DAB. Using ImageJ, the extent of BBB damage was quantified as the percentage of area of IgG positive staining per ipsilateral hemisphere. For Texas Red dextran perfusion, mice were deeply anesthetized with isoflurane, and Texas Red dextran 70 kDa (D1864, Invitrogen) in PBS (50 mg/ml) was injected into the inferior vena cava for 2 minutes of circulation. Mice were immediately killed by decapitation. Brains were quickly extracted and post-fixed in 4% PFA for 24 hours then cryoprotected by 30% sucrose for 48 hours. After O.C.T. embedding, brains were cut coronally in 20-μm sections. Slices were directly coverslipped using mounting media with 4′,6-diamidino-2-phenylindole (DAPI) (H-1200, Vector Laboratories).

Flank tumors and whole brains were fixed and processed for immunohistochemistry as described previously.[4 (link)] Tissues were incubated with primary antibody at room temperature for 1 hr. Rabbit polyclonal anti-cleaved Caspase 3 antibody (Cell Signaling) 1:500, and anti-phospho Histone H3 (Ser 10) (Cell Signaling) 1:200 were used. TBS-T was used for washes. Detection was performed with avidin-biotin-horseradish peroxidase complex (ABC; Vector Laboratories) followed by diaminobenzidine as the chromogen. Nuclei were counterstained with hematoxylin. Slides were visualized on an Olympus 1X37 light microscope. Cells were counted in 20X fields to greater than 1000 cells per specimen.