Anti pkd1

Animals were deeply anesthetized and perfused transcardially with physiological saline followed by 4% paraformaldehyde at a speed of about 0.7 mL per hour using a peristaltic pump (RP-2000; EYELA). The intracranial arteries were isolated under the microscope and embedded in optimum cutting temperature compound (Tissue-Tek; Sakura Fine Technical Co, Tokyo, Japan). Serial 8 μm sections were cut and mounted on silane-coated slides. For immunofluorescence staining, tissue samples were washed in PBS, blocked with 5% donkey serum in PBST (0.1% Triton-X in PBS), then incubated with primary antibodies followed by the appropriate fluorescent-labeled secondary antibodies. The following primary antibodies were used: anti-VE-cadherin (BD Biosciences, Franklin Lakes, NJ; 1:100), anti-Claudin 1 (Cell Signaling, Danvers, Mass; 1:100), anti-PKD1 (Sigma, 1:200), anti-PKD2 (Sigma, 1:200), anti-acetylated α-tubulin (Sigma, 1:1,000). Secondary antibodies for immunofluorescence staining are as follows: Alexa488-, Alexa594-conjugated IgG antibodies (Invitrogen, Waltham, Mass; 1:1,000). All specimens were counterstained with DAPI (Invitrogen, 1:1,000) and finally mounted in Vectashield (Vector Laboratories, Burlingame, Calif). Immunofluorescence images were obtained using a laser scanning microscope 780 confocal microscope (Zeiss).