Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center
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.
- Paraffin sections are placed at 60oC 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
- Antigen retrieval, pH 6.0 (times will vary according to microwave).
- 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).
- Microwave according to instructions on microwave.
- Cool on countertop, 15 min.
- Rinse with dH2O
- 1X PBS, 5 min.<