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 paraffin sections of human lung  from the HTC for NKX2.1, TP63 and KRT5 with antigen retrieval.

Day 1

1. Paraffin sections are placed on slide warmer for 1 hour 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 96^oC. *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 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 and incubate on tissue overnight @ 4^oC.

Day 2

1. Rinse slides in PBS-T 3X, 5 min.
2. Goat Alexa Fluor 488 anti-mouse IgG (A11001, Lot# 1298479, for anti-TP63) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, for anti-NKX2.1) at 1:200, Goat Alexa Fluor 633 anti-rabbit IgG (A21070, Lot # 1668691, anti-KRT5) at 1:200, in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
3. Rinse in PBS-T 3X, 5 min.
4. Dilute DAPI 1:2000 and apply to slides for 10 min.
5. Wash in PBS-T 3X, 5 min.
6. Rinse slides in 0.1M PB or TB, 3X, 5 min.
7. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
8. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
9. Allow Prolong Gold to cure overnight at room temp in light sealed box.

Tissue Used:
LMH-15-D007-RLL-10B2-14
Age: 20 Month Old
Gender: Male
Race: African American

Appendix
Antigen Retrieval Solution

18ml Solution A- 0.1M Citric Acid (Sigma, C1909) pH 2.5

82ml Reagent B- 0.1M Sodium Citrate (Sigma, S4641 ) pH 8.2

1L dH₂O

pH 6.0