Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center

Pryhuber_Lab

 

Gene
Nkx2-1 LungGens Ensemble Genecards NCBI The Human Protein Atlas
Pecam1 LungGens Ensemble Genecards NCBI The Human Protein Atlas
Vim LungGens Ensemble Genecards NCBI The Human Protein Atlas

 

Immunofluorescence- PECAM-1, VIM, NKX2.1

 

Purpose:  Stain slides of 7µm frozen sections of postnatal human lung from HTC for PECAM-1, VIM, NKX2.1 with antigen retrieval.

Day 1

  1. For Frozen tissue, rinse 2X in PBS, 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 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).
  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 mouse anti-PECAM-1 (BBA7, R&D Systems) dilute 1:100, for goat anti- VIM (SC7557, Lot # C1215) dilute 1:100, For Rabbit anti-NKX2.1 (RB TTF-1 1231, Lot# 0912A) dilute 1:500 in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated O/N @ 4oC.

Day 2

  1. Rinse slides in PBS-T 3X, 5 min.
  2. Apply secondary antibody , Donkey Alexa Fluor 488 anti-mouse IgG (A21202, lot# 1562298, anti-PECAM-1), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, anti-VIM ) at 1:200, Donkey Alexa Fluor 647 anti-rabbit IgG (A31573, anti-1) 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.
  10. Store slides in a light-sealed box at 4oC.

 

Tissue Used:   

LMH-15-D032-LLL-8B2.9

Gender: Male

Age: 3 Years

Gestational Age at Birth:  36 weeks

 

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