Postnatal Day 10 CALCA, SCGB1A1, and TUBA1A Imaging

Postnatal Day 10 CALCA, SCGB1A1, and TUBA1A Imaging

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



Immunofluorescence CALCA, SCGB1A1, TUBA1A1


Purpose: Stain slides of 7µm frozen sections of PND10 C57Bl6 lungs for CALCASCGB1A1, TUBA1A1 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. Briefly equilibrate slides in Antigen Retrieval Buffer.
  2. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 90oC. *We usually do a series of three runs to equal the time/temp because of evaporation (refill coplin jars with dH2O).
  3. Microwave according to instructions on microwave.
  4. Cool on countertop, 15 min.
  5. Rinse with dH2O
  6. 1X PBS, 5 min.
  7. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  8. For mouse anti –CALCA (C7113, Sigma) dilute 1:100 in blocking buffer, For guinea pig anti-CCSP (G210, Bl. 3/24/11) dilute 1:1500, for mouse anti- TUBA1A1(T7451, Sigma) dilute 1:1000 in blocking buffer. Spin for 10 minutes in a µfuge, 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-mouse IgG1 (A21121, Lot# 1345050, for anti-CALCA ) dilute 1:200, Goat Alexa Fluor 555 anti-guinea pig IgG (A21435, Lot# 1571699, CCSP) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG2B (A21242, Lot# 1148959, anti-TUBA1A1) at 1:200, in blocking buffer. Vortex and µfuge at full speed for 10 minutes. 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 light sealed box at 4o


Tissue Used:
PND10 C57BL6            
Gender: Male
Weight= 4.1g


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