Postnatal Day 10 C57BL6 HOPX, SFTPC, and ACTA2 Confocal Imaging

Immunofluorescence- HOPX, SFTPC, and ACTA2

 

Purpose: Stain slides of 5µm paraffin sections of C57BL/6J PND10 lungs for HOPX, SFTPC, and ACTA2 with antigen retrieval.

Day 1 

  1. For Frozen tissue remove OCT and rehydrate by washing in 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS. Briefly equilibrate slides in Antigen Retrieval Buffer.
  2. Briefly equilibrate slides in Antigen Retrieval Buffer.
  3. 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).
  4. Microwave according to instructions on microwave.
  5. Cool on countertop, 15 min.
  6. Rinse with dH2O
  7. 1X PBS, 5 min.
  8. Block in 4% Donkey serum/PBS-T, 2 hours at RT.
  9. For Rabbit anti-HOPX (SC-30216, Lot # C2212, Santa Cruz) dilute 1:200, For goat anti-SFTPC (SC-7706, Santa Cruz) dilute 1:100, for mouse anti- ACTA2 (A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated O/N @ 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# 1387792, for anti-HOPX), Donkey Alexa Fluor 568 anti-goat IgG (A11057, Lot# 1235787, anti- SFTPC) at 1:200, Donkey Alexa Fluor 647 anti-mouse IgG ( A31571, Lot# 1366514, anti- ACTA2) at 1:200, in blocking buffer. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  3. Spin down in µfuge for 10 min, apply to tissue and incubated at room temperature for 1 hour.
  4. Rinse in PBS-T 3X, 5 min.
  5. Dilute DAPI 1:2000 and apply to slides for 15 min.
  6. Wash in PBS-T 3X, 5 min.
  7. Rinse slides in 0.1M PB or TB, 3X, 5 min.
  8. Add 1 drop of Prolong Gold anti-fade mounting medium (P36930).
  9. Coverslip with Gold Seal Coverslip (Cat# 3422 Electron Microscopy Sciences, 22 X 22 mm).
  10. Allow Prolong Gold to cure overnight at room temp in light sealed box.
  11. Store slides in a light sealed box @ 4oC.

Tissue Used:   

LMM.16.84A.5L.46

PND10 C57BL6

Gender: Male

Weight= 4.5g

 

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