Adult Human Lung Immunofluorescence and Confocal Imaging Showing Expression of NKX2.1, VIM, and ACTA2

   Donor tissue kindly provided by Dr. Scott Randell, University of North Carolina

Immunofluorescence-NKX2.1, VIM and ACTA2

Purpose:  Stain slides of 7µm frozen sections of human lung for NKX2.1, VIM and ACTA2 with antigen retrieval.

Day 1

1. For Frozen tissue, rinse 2X in PBS to rehydrate tissue, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
2. Briefly equilibrate slides in Antigen Retrieval Buffer.
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 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).
5. Microwave according to instructions on microwave.
6. Cool on countertop, 15 min.
7. Rinse with dH₂O.
8. 1X PBS, 5 min.
9. Block in 4% Donkey serum/PBS-T, 2 hours at RT.
10. For Rabbit anti-NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, for goat anti- VIM (SC7557, Lot # C1215) dilute 1:100, for mouse anti- ACTA2 (A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated O/N @ 4^oC.

Day 2

1. Rinse slides in PBS-T 3X, 5 min.
2. Apply secondary antibody , Donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1387792, for anti-1), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, anti-VIM ) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG A31571, Lot# 1366514, anti- ACTA2) 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:

LMHA-UNC_DD036L-04HF-1-24
Age: 47 years
Gender: Male
Race: Caucasian
Non-smoker

LMHA-UNC_DD037L-05HF-1-23 
Age: 43 years
Gender: Female
Race: Caucasian
Non-smoker

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