Differentiation of Mature and Immature Vasculature

protocol.md

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.