Postnatal Day 10 C57BL6 SCGB1A1, NKX2.1, and TUBA1A Confocal Imaging

Immunofluorescence- NKX2.1, SCGB1A1,  TUBA1A

Purpose: Stain slides of 7µm frozen sections of C57BL/6J PND10 NKX2.1, SCGB1A1and TUBA1A 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. Antigen retrieval, pH 6.0 (times will vary according to microwave). Briefly equilibrate slides in Antigen Retrieval Buffer.
  3. 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).
  4. Microwave according to instructions on microwave.
  5. Cool on counter top, 15 min.
  6. Rinse with dH2O.
  7. 1X PBS, 5 min.
  8. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  9. For Rabbit anti-NKX2.1 (R1231) dilute 1:1000. For guinea pig anti-SCGB1A1 (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti- TUBA1A (T7451, Sigma) dilute 1:1000 in blocking buffer. Spin down in µfuge for 10 min, apply to tissue 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 anti-1), Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 123