Wittrup:Immunohistochemistry of paraffin embedded sections

Immunohistochemistry of paraffin embedded 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)

250 ml of 1% H2O2 (8.3 ml of 30% 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 1

1. Dewax slides. Load slides into black holder in the hood and place holder into the machine. Press Program->8) dewax. It takes 15 mins. After dewaxing, press C to acknowledge.

2. Antigen unmasking. Fill box with 250 ml 10mM sodium citrate buffer and bring to histology room. Unload slides into citrate buffer. Heat in microwave for 1 min at power 10, then 9 mins at power 5.

3. Cool slides for 20 mins at room temp.

4. Wash with water 3x 5mins.

5. Endogenous peroxidase blocking. Incubate slides for 10 mins at room temperature.

6. Wash with water 3x 5mins.

7. Wash in 1x PBS for 5 mins.

8. Blocking. Mark tissue with a pap pen. 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.

9. Primary antibody incubation. Dilute antibody in blocking solution (usually 1:100) and incubate overnight at 4C.

Day 2

1. Bring slides from 4C and let it equilibrate to RT for 20-30mins.

2. Wash slides in 1xPBS 3x5mins. Cover sample with enough PBS and remove PBS by aspiration after 5 mins.

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

4. 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.

5. Wash in 1X PBS 3x5mins

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

7. Wash with 1X PBS 3x5mins.

8. 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.

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

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

11. Transfer slides into hood. Be careful not to trip 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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