Histology

scientificprotocols authored almost 4 years ago

Materials Needed

  1. PAP pen (Abcam ab2601)
  2. Histochoice MB Fixative (Electron Microscopy Sciences 64115-01)
  3. 10xPBS (Fisher BP3994)
  4. Triton X (Roche 12754423)
  5. Bovine serum albumin (Sigma A8022-500G)
  6. Mouse CD31 PECAM
  7. Rabbit alpha smooth muscle actin (Abcam ab5694)
  8. Alexa Fluor 555 donkey anti-mouse IgG (H L) (Invitrogen A31570)
  9. Alexa Fluor 488 donkey anti-rabbit IgG (H L) (Invitrogen A21206)

Solutions

  1. Wash Buffer: 1xPBS
  2. 0.2% Triton-X: Add 20 µl of Triton-X to 10 mL of 1xPBS
  3. Block Buffer: Add 0.1 g of BSA to 10 mL of 1xPBS
  4. Primary and secondary antibodies: Add 20 µl of antibody to 10 mL of block buffer

Proceedure

  1. Encircle sections with PAP pen.
  2. Fix in fixative for 45 minutes.
  3. Wash once with wash buffer for 10 minutes.
  4. Permeablize with 0.2% Triton-X for 20 minutes
  5. Wash three times with wash buffer for 5 minutes each.
  6. Incubate with primary antibodies for 1 hour at room temperature.
  7. Wash three times with wash buffer for 5 minutes each.
  8. Incubate with secondary antibodies for 1 hour at room temperature in dark.
  9. Wash three times with wash buffer for 5 minutes each.
  10. Mount with DAPI and let dry.

DOI

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