Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center
Immunofluorescence-NKX2.1, TP63 and KRT5
Purpose: Immunofluorescence on slides of human lung from the HTC for NKX2.1, TP63 and KRT5 with antigen retrieval.
- Paraffin sections are placed on slide warmer for 1 hour 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.
- Briefly equilibrate slides in Antigen Retrieval Buffer.
- 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.
- Block in 4% Goat serum/PBS-T, 2 hours at RT.
- For mouse anti-TP63 (CM163A, lot # 102214, Biocare) dilute 1:100, For guinea pig anti-NKX2.1 (G237, Bl. 8/25/11) dilute 1:200 for rabbit anti-KRT5 (PRB-160P-200, Lot# D13DF01179, Biolegend) Dilute 1:100 in blocking buffer. Spin down in µfuge for 10 minutes a