14 Month-Old Human Lung Immunofluorescence and Confocal Imaging Donor D072 Showing Expression of FN1, ABCA3, and ACTA2

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

Pryhuber_Lab 

 Immunofluorescence- FN1, ABCA3, and ACTA2

 

 

Purpose:  Stain slides of 7µm frozen sections of 3 year-old human lung provided by the HTC for FN1, ABCA3, and ACTA2 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 rabbit anti-FN1 (AB2413, ABCAM, anti-Fibronectin), for mouse anti-ABCA3 (WMAB-17G524, Seven Hills Bioreagents) 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.

 

Day 2

  1. Rinse slides in PBS-T 3X, 5 min.
  2. Apply secondary antibody, donkey Alexa Fluor 488 anti-rabbit IgG (A21206, Lot# 1608521, anti-FN1), Goat Alexa Fluor 568 anti-mouse pig IgG2B (A21144, Lot# 1584300, for anti-ABCA3) 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 20 min, apply to tissue and incubated at room temperature for 1 hour.
  3. Wash 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 light sealed box @ 4oC.

 

Tissue Used:
LMH-18-D072-LLL-12B2.19
Gender: Female
Age: 14 Months

 

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

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