6 Day-Old Human Lung NKX2.1, KI67, and ACTA2 Confocal Imaging
Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center
- Paraffin sections are placed at 60oC 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 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 Rabbit anti-NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:1000, For mouse anti-Ki-67 (55603, BD Biosciences, Lot# 6251774) dilute 1:100, for mouse anti-ACTA2 (α Smooth muscle actin, A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in µfuge for 10 minutes and incubate on tissue overnight @ 4oC.
- Rinse slides in PBS-T 3X, 5 min.
- Apply secondary antibody, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for anti-NKX2.1), Goat Alexa Fluor 568 anti-mouse IgG1 (A21124, Lot# 1600878, for anti-Ki-67) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A (A21136, Lot# 1345055, 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.
- Rinse in PBS-T 5X.c
- Dilute DAPI 1:2000 and apply to slides for 10 min.
- Wash in PBS-T 3X, 5 min.
- Rinse slides in 0.1M PB or TB, 3X, 5 min.
- Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
- Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
- Allow Prolong Gold to cure overnight at room temp in light sealed box.
- Store in light-sealed box @ 4oC.
Age: 6 Days
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