Differentiation of Mature and Immature Vasculature

### Materials Needed 1. PAP pen (Abcam ab2601) - Histochoice MB Fixative (Electron Microscopy Sciences 64115-01) - 10xPBS (Fisher BP3994) - Triton X (Roche 12754423) - Bovine serum albumin (Sigma A8022-500G) - Mouse CD31 [PECAM] (BD Biosciences 555025) - Rabbit alpha smooth muscle actin (Abcam ab5694) - Alexa Fluor 555 donkey anti-mouse IgG (H L) (Invitrogen A31570) - Alexa Fluor 488 donkey anti-rabbit IgG (H L) (Invitrogen A21206) ### Solutions 1. Wash Buffer: 1xPBS - 0.2% Triton-X: Add 20 µl of Triton-X to 10 mL of 1xPBS - Block Buffer: Add 0.1 g of BSA to 10 mL of 1xPBS - Primary and secondary antibodies: Add 20 µl of antibody to 10 mL of block buffer ### Proceedure 1. Encircle sections with PAP pen. - Fix in fixative for 45 minutes. - Wash once with wash buffer for 10 minutes. - Permeablize with 0.2% Triton-X for 20 minutes - Wash three times with wash buffer for 5 minutes each. - Incubate with primary antibodies for 1 hour at room temperature. - Wash three times with wash buffer for 5 minutes each. - Incubate with secondary antibodies for 1 hour at room temperature in dark. - Wash three times with wash buffer for 5 minutes each. - Mount with DAPI and let dry.