Calca LungGens Ensemble Genecards NCBI The Human Protein Atlas
Scgb1a1a LungGens Ensemble
Genecards NCBI The Human Protein Atlas
Tuba1a LungGens Ensemble Genecards NCBI The Human Protein Atlas


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.
  1. 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).
  2. Microwave according to instructions on microwave.
  3. Cool on countertop, 15 min.
  4. Rinse with dH2O
  5. 1X PBS, 5 min.
  6. Block in 4% Goat serum/PBS-T, 2 hours at RT.
  7. For mouse anti –CALCA (C7113, Sigma) dilute 1:100 in blocking buffer, 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 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:    LMM.15.66A.6.73

PND01 C57BL6

Gender: Female

Weight= 1.14g