Human Lung Confocal Imaging Donor D034 showing expression of HSPA5, ABCA3, and ACTA2

Purpose:  Stain slides of 5µm frozen sections of human lung from HTC and from Wambach/ ABCA3 mutations for HSPA5, ABCA3, and 
ACTA2 with antigen retrieval.

Day 1

1. Paraffin sections are placed at 60^oC for 2 hours to overnight to melt paraffin.
2. 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.
3. Antigen retrieval, pH 6.0 (times will vary according to microwave).
4. 10 mM sodium citrate, pH 6.0, and heat in a microwave at 96^oC. *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).
5. Microwave according to instructions on microwave.
6. Cool on countertop, 15 min.
7. Rinse with dH₂O
8. 1X PBS, 5 min.
9. Block in 4% Donkey serum/PBS-T, 2 hours at RT.
10. For Rabbit HSPA5 (GRP78/BiP, G9043, Sigma) dilute 1:100, for mouse anti-ABCA3 (WMAB-17G524, Seven Hills Bioreagents)
    dilute 1:100, for mouse anti-ACTA2 (α Smooth muscle actin, A5228, Sigma) dilute 1:2000 in blocking buffer. Spin down in
    µfuge for 10 minutes and incubate on tissue overnight @ 4^oC.

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-HSPA5), Goat Alexa Fluor
   568 anti-mouse pig IgG2B (A21144, Lot# 1584300, for anti-ABCA3) at 1:200, Donkey Alexa Fluor 633 anti-mouse IgG2A 
   A21136, Lot# 1345055, 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. Wash 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 @ 4^oC

Tissue Used:
LMH-16-D034-RLL-7B2.12
Age: 6 Months Old
Gender: Male

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