Postnatal Day 1 C57BL6 HOPX, AGER, and ACTA2 Confocal Imaging

Immunofluorescence-  HOPX, AGER, and ACTA2

Purpose:  Stain slides of 7µm frozen sections of C57BL6/J E18.5 lungs for HOPX, AGER, and ACTA2 with antigen retrieval.

 

Day 1

  1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.
  3. Antigen retrieval, pH 6.0 (times will vary according to microwave).
  4. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96 o*We usually do a series of three runs (6-7 minutes each run) to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  5. Microwave according to instructions on microwave.
  6. Cool on countertop, 15 min.
  7. Rinse with dH 2O
  8. 1X PBS, 5 min.
  9. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  10. For rabbit anti- HOPX (SC-30216, Lot # C2212, Santa Cruz) dilute 1:200, For rat anti-AGER ( MAB 1179-500, RND Systems) dilute 1:100, for mouse anti- ACTA2 ( α Smooth muscle actin, A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down