4 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor 075 Showing Expression of HOPX, ABCA3, and SFTPC

Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center

Pryhuber_Lab

Immunofluorescence- HOPX, ABCA3, and SFTPC

 

Purpose:  Stain slides of 5µm paraffin sections of human lung from the HTC for HOPX, ABCA3, and SFTPC with antigen retrieval.

Day 1

  1. Paraffin sections are placed at 60oC for 2 hours to overnight to melt paraffin.
  2. 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.
  3. Briefly equilibrate slides in Antigen Retrieval Buffer.
  4. Antigen retrieval, pH 6.0 (times will vary according to microwave).
  5. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96oC. *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).
  6. Microwave according to instructions on microwave.
  7. Cool on countertop, 15 min.
  8. Rinse with dH2O
  9. 1X PBS, 5 min.
  10. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  11. For Rabbit anti-HOPX (SC-30216, Lot# C2212, Santa Cruz) dilute 1:100, for