E16.5 C57BL6 HOPX, SFTPC, and ACTA2 Confocal Imaging


Immunofluorescence- HOPX, SFTPC, and ACTA2


Purpose: Stain slides of 5µm paraffin sections of C57BL/6J E16.5 lungs for HOPX, SFTPC, and ACTA2 with antigen retrieval.

Day 1

  1. Paraffin sections are placed at 60 oC for 2 hours to overnight to melt paraffin. Paraffin sections are then placed in xylene 3X for 10 minutes each, followed by 3X in 100% ETOH for 3 minutes each, 95% ETOH for 3 minutes each, and 70% ETOH for 3 minutes each. Slides are then placed in 1X PBS for 5 minutes to completely rehydrate tissue.
  2. For Frozen tissue remove OCT and rehydrate by washing in 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
  3. Briefly equilibrate slides in Antigen Retrieval Buffer.
  4. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 96 oC. *We usually do a series of three runs 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% Donkey serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti- HOPX (SC-30216, Lot # C2212, Santa Cruz) dilute 1:200, For goat anti-SFTPC (SC-7706, Santa Cruz) dilute 1:100, for mouse anti-  ACTA2 (A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated O/N @ 4o C.
Day 2
  1. Rinse slides in PBS-T 3X, 5 min.
  2. Apply secondary antibody , Donkey Alexa Fluor 488 anti-rabbit IgG ( A21206, Lot# 1387792, for anti-HOPX