E16.5 C57BL6 SOX9, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence- SOX9, NKX2.1 and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57BL6 E16.5 lungs for SOX9, NKX2.1 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% Goat serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti-SOX9 (AB-5535, Lot # 2167153, Millipore) dilute 1:100, For guinea pig anti-NKX2.1 (G237, Bl. 10/7/11) dilute 1:500, for mouse anti- ACTA2 (A5228, α Smooth muscle actin, Sigma) dilute 1:2000 in blocking buffer. Spin down antibody cocktail in a µfuge for 10 min and incubate on tissue overnight @ 4oC.

Day 2

  1. Rinse slides in PBS-T 3X, 5 min.
  2. Apply secondary antibody, Goat Alexa Fluor 488 anti-rabbit IgG (A11034, Lot# 1298480, for SOX9), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, GPTTF-1) at 1:200 at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG (A21136, Lot# 1345055, anti-ACTA2) at 1:200, in blocking buffer. Spin down antibody cocktail in a µfuge for 10 min 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 @ 4oC

Tissue Used:

LMM.14.24.4.34 E16.5

Gender: Male

Crown-to-Rump Length: 16.0 mm

Weight=0.592g

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