E18.5 C57BL6 HOPX, SFTPC, and ACTA2 Confocal Imaging

Immunofluorescence- HOPX, SFTPC, and ACTA2

 

Purpose: Stain slides of 5µm paraffin sections of C57BL/6J E18.5 lungs for HOPX, SFTPC, and ACTA2 with antigen retrieval.

Day 1
 
  1. Paraffin sections are placed at 60 oC for 2 hours to overnight to melt paraffin. Paraffin sections are then placed in xylene 3X for 10 minutes each, followed by 3X in 100% ETOH for 3 minutes each, 95% ETOH for 3 minutes each, and 70% ETOH for 3 minutes each. Slides are then placed in 1X PBS for 5 minutes to completely rehydrate tissue.
  2. 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.
  3. Briefly equilibrate slides in Antigen Retrieval Buffer.
  4. 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 96 oC. *We usually do a series of three runs 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 dH 2O
  8. 1X PBS, 5 min.
  9. Block in 4% Donkey serum/PBS-T, 2 hours at RT.
  10. 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 @ 4o C.
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. Rinse in PBS-T 3X, 5 min.
  4. Dilute DAPI 1:2000 and apply to slides for 15 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 a light sealed box @ 4o C.
 
Tissue Used:

LMM.14.49.5.55 E18.5

Gender: Male

Crown-to-Rump Length: 22.0 mm

Weight=1.17g

 
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 dH2 O
pH 6.0