Laminin Isoforms and Tight Junction Proteins in Brain

Cortex and striatum were carefully dissected and immediately homogenized on ice. Total protein concentration was determined using the BCA Protein Assay Kit (Pierce 23,227). Equal amount of protein was loaded and separated in SDS-PAGE and transferred to PVDF membranes (Millipore). Next, the membranes were probed with primary antibodies [anti-Laminin-α2 (1:500, Sigma L0663), anti-Laminin α5 (1:800, generated as described in [47 (link)]), Claudin-5 (1:500, ThermoFisher 35–2500), ZO-1 (1:500, ThermoFisher 61–7300), and anti-GAPDH (1:1000, Abcam AB9484)] over night at 4 °C, followed by appropriate horseradish peroxidase-conjugated secondary antibodies [donkey anti-mouse (1:2500, Jackson ImmunoResearch Laboratory 715–035-151), donkey anti-rabbit (1:2500, Jackson ImmunoResearch Laboratory 711–035-152), and donkey anti-rat (1:2500, Jackson ImmunoResearch Laboratory 712–035-153] at room temperature for 1 h. Then, target proteins were visualized using the ChemiDoc Imaging System (Bio-Rad). For quantification, the density of target blots was normalized to that of GAPDH, and the expression of target proteins in α5-PKO brains was normalized to that in control brains. Four animals were used for quantification.