Wittrup:Immunohistochemistry of frozen sections

Immunohistochemistry of frozen sections

Materials

primary antibody

biotin-conjugated secondary antibody

serum in which secondary antibody is raised

250 ml of 10mM sodium citrate pH6 (dilute from 0.1M stock)

1ml 0.3% H2O2

5ml 0.1% tween20 in PBS

DAB substrate kit for peroxidase (Vector laboratories #SK-4100)

Vectastain ABC kit (Vector laboratories)

Hydrophobic slide marker

Humidifier chamber (place a piece of filter paper inside a pipette tip box and fill the bottom of box with water)

Day 0

1. Take out slides and dry overnight.

Day 1

1. Draw a circle around specimen with Pap Pen. Dry for 2 min.

2. Wash in 1x PBS for 5 mins.

3. Blocking. Incubate with 5% serum in 1x PBS for 1 hr at R.T. Add enough serum to cover sample. 1ml is enough for 4 samples. Add 4 drops of avidin/ml serum

4. Rinse 3x2mins

5. Primary antibody incubation. Dilute antibody in blocking solution (usually 1:100), add 4 drops of biotin/ml. Incubate overnight at 4C.

Day 2

1. Bring slides from 4C and let it equilibrate to RT for 20-30mins. 2. Wash slides in PBS 3 x 2mins. Cover sample with enough PBS and remove PBS by aspiration.

3. Fix section in ice cold 1% paraformaldehyde for 5 mins.

4. Rinse in PBS 3x2 mins.

5. Block with 0.1% H2O2 for 10 mins and rinse in PBS 3x2 mins.

6. Dilute secondary antibody in blocking solution (1:200) and incubate for 1 hr at R.T.

7. Meanwhile, make up ABC and let it sit for 30 mins before use. 5ml 0.1% tween20 in PBS+1 drop of A+ 1 drop of B. It is enough for 8-10 samples.

8. Wash in 1X PBS 3x2mins

9. Add ABC for 30 mins at R.T.

10. Wash with 1X PBS 3x2mins

11. Make DAB solution. 920 μl water + 20 μl pH7.5 buffer + 1 drop of DAB +20 μl H2O2. Vortex. Add DAB solution until slides change color. Adding more drops of DAB decreases the intensity.

12. Transfer to 1X PBS to rinse DAB off.

13. Counterstain with hematoxylin (HCS Erica). Samples are in xylene.

14. Transfer slides into hood. Be careful not to drip xylene onto the floor. In the hood, align and clamp blue color slide holder. Transfer slides from black slide holder into blue slide holder. Be sure to orient slides correctly (slides facing down when stick is up). Mount cover slip using mounting machine (only for samples in xylene). Remember to place the dispenser back to its original position.

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