Yun Jiang and Huang-Tian Yang

Procedures for immunofluorescence staining

The heart sections are put in a fume hood for 5 minutes
Wash sections in 1x PBS for 5 min (3 times total)
Immobilize sections in 4% paraformaldehyde (4% PFA) for 20 min at room temperature after the staining area of tissues is marked by a block pen
Wash sections in 1x PBS for 5 min at room temperature (3 times in total)
Remove PBS and add permeabilization buffer (0.4%-Triton in 1x PBS) for 20 min at room temperature
Wash sections in 1x PBS for 5 min at room temperature (3 times total)
Remove PBS and add block buffer for 1 hours at room temperature
Remove block buffer and add primary antibodies in antibody diluent solution overnight at 4℃
Remove primary antibodies and wash sections with 1x PBS for 5 min (3 times total) at room temperature
Remove PBS and add species-specific Alexa fluorophore-conjugated secondary antibodies in antibody diluent solution for 1 hours at room temperature (keep in dark)
Remove secondary antibodies and wash sections with 1x PBS for 5 min (3 times total) at room temperature
Remove secondary antibodies and stain with a nuclear marker for 5 min at room temperature
Wash sections in 1x PBS for 5 min at room temperature (3 times total)
Gently shake to let the liquid on the samples dry
Dispense one or two drops of Fluorescent Mounting Medium (DAKO) onto staining areas in sections, apply glass coverslip over Fluorescent Mounting Medium, and allow the medium to spread evenly over the specimen.
Sections are examined under a fluorescent a microscope which should to be done within two weeks.

Solutions:

1. Permeabilization buffer (0.4%-Triton, in 1x PBS)

2. Block buffer (5% bovine albumin, in 1x PBS)

3. Antibody diluent solution (3% bovine albumin, in 1x PBS)

Tested primary antibodies:

Antibody	Concentrations	Company	Catalogue Number
CD31	1:200	Abcam,	ab28364
a-SMA	1:400	Abcam	ab5694
cTnT	1:500	Abcam	ab8295

Note: cTnT, cardiac troponin T; α-SMA, alpha-smooth muscle actin

Image taken from a heart section staining with α-SMA (Green) and cTnT (Red).

20191021-IR-Sr-4W-816-CD31-cTnT-20X-1_c1+3

Image taken from a heart section staining with CD31 (Red).