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Human Lung Confocal Imaging Donor D034 showing expression of HSPA5, ABCA3, and ACTA2

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

Pryhuber_Lab

Immunofluorescence- HSPA5, ABCA3, and ACTA2 

Purpose:  Stain slides of 5µm frozen sections of human lung from HTC and from Wambach/ ABCA3 mutations for HSPA5, ABCA3, and 
ACTA2 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. 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 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).
  5. Microwave according to instructions on microwave.
  6. Cool on countertop, 15 min.
  7. Rinse with dH2O
  8. 1X PBS, 5 min.<