Immunohistochemistry of fresh frozen mouse heart sections

Tissue preparation

1. Remove heart from euthanized mouse.
2. Blot out blood and wrap heart in aluminum foil.
3. Freeze organ by dipping in liquid nitrogen or by placing on dry ice.
4. Store the heart in a 1.5ml eppendorf tube, tightly closed and keep at -80º C until sectioning on a cryostat.

 

Cryostat sectioning:

1. The cryostat chamber should be set to -25º C to -27º C.
2. Pre-arrange in a holder and chill on crushed ice super-frost slides.
3. Thaw mounts on slides, 5-6 10 µm thick transverse sections per slides.
4. Arrange slides in a box and keep at -80º C until fixation for immunohistochemistry.

 

Fixation:

1. Place a volume of acetone and a coplin jar in advance in a -18º C freezer.
2. Remove slides from -80º C and place leaning in a flat tray in a fume hood to dry at room temperature for 10 minutes.
3. Place slides in the pre-chilled coplin jar and pour pre-chilled acetone in jar to cover the slides.
4. Place coplin jar in -18º C freezer for 10 min.
5. Pour out acetone and allow slides to dry in a fume hood for 10 minutes.
6. Fill jar with 0.02 M phosphate buffered saline (PBS) and put in refrigerator (2-8º C) until immunohistochemical processing.

 

Antigen retrieval (hydrogen peroxide):

1. Remove slides from coplin jar and put on a flat tray and add 200 µl of antigen retrieval solution on top of each slide.
2. Cover slide with a rectangle of parafilm and leave at room temperature for 25 minutes.
3. Rinse off antigen retrieval solution with PBS and arrange slides leaning in a box to dry for 5 minutes.

 

Hydrogen peroxide antigen retrieval solution

Component

Percentage of final volume

0.2 M dibasic phosphate

37.4

0.2 M monobasic phosphate

12.5

Methanol

20

Triton 30%

0.2

Hydrogen peroxide 30%

5

Distilled deionized water

25

 

Staining procedure

1. Arrange slides on a flat tray.
2. Add 200 µl primary antibody solution on each slide and cover with parafilm.
3. Incubate at room temperature for 1h and then refrigerate overnight.
4. Arrange slides on a flat tray and incubate with the secondary antibody solution for 2 hrs at room temperature.

 

Primary antibody solution

Component

Percentage of final volume

0.02 M Phosphate buffered saline

97

Normal serum (species of second antibody)

2

Triton 30%

0.2

Tween 20%

1

Primary antibody

To be determined by end user

 

Secondary antibody solution

Component

Percentage of final volume

0.02 M Phosphate buffered saline

97

Normal serum (species of second antibody)

2

Triton 30%

0.2

Tween 20%

1

Secondary antibody conjugated to fluorophore or biotin*

To be determined by end user

*If the secondary antibody is conjugated to biotin, it should be next incubated with a solution of streptavidin conjugated with a fluorophore for 1 hr at room temperature.

 

Detection procedure using fluorescent microscopy

Counter-staining, and cover-slipping:

1. Stain section on slides with DAPI (Molecular Probes), by placing DAPI solution on each slide, and covering the slide with a piece of parafilm to spread the solution evenly on the slide.
2. After 2 minutes, remove the parafilm and rinse the slide with 0.5 ml distilled deionized water using a pipette (repeat twice).
3. Let slides dry in a fume hood for 0.5 h.
4. Coverslip using the glue ImmuMount (Shandon, UK).
5. Leave slides to dry overnight in the dark.
6. Store at -18º C until confocal microscopy.