Figure 1
From: Identification of lamprey variable lymphocyte receptors that target the brain vasculature
Brain microvessel plasma membrane (BMPM) isolation and lamprey immunization validation. (A) Isolated mouse brain microvessels were stained with trypan blue. Scale bar = 25 μm. (B) Cropped western blots for Glut-1 and GFAP along with normalized GGT activity for the various fractions generated during BMPM isolation. Raw western blot data can be found in Fig. S11. Hypotonic lysis was used to disrupt the isolated brain microvessels. Following centrifugation, the supernatant (HLS) was separated from the lysed microvessel fragments. Sonication of the microvessel fragments and centrifugation separated the plasma membranes (PM) from the basement membrane pellet (BM). (C) Pooled plasma from BBB immunized lampreys or a plasma sample from a naïve lamprey were used to immunolabel mouse brain sections (red) and brains were counterstained with fluorescent IB4-lectin (green) to identify brain microvessels. Scale bar = 50 μm. (D) Glycan microarray analysis. Plasma samples from a naïve lamprey (Naïve, gray), lamprey immunized with human erythrocytes (RBC, blue), or BMPM immunized lamprey (BBB, red) were used to probe the CFGv5.2 glycan microarray. RFU = relative fluorescence units.
