Postnatal Day 7 C57BL6 ARL13B, NKX2.1, and ACTA2 Confocal Imaging

Immunofluorescence-ARL13B, NKX2.1, and ACTA2

 

Purpose:  Immunofluorescence on slides of 7µm frozen sections of C57Bl/6 PND7 lungs for ARL13B, 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% Donkey serum/PBS-T, 2 hours at RT.
  10. For Rabbit anti-Arl13b (17711-AP,Proteintech) dilute 1:200, For goat anti-EMCN (Endomucin, AF4666, R&D Systems) dilute 1:200, 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, for anti-Arl13b), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1485187, for anti- EMCN) at 1:200, Donkey Alexa F