Immunohistochemistry protocols

Deparaffinization

1. Transferring to the xylene tank, replace the xylene twice every 5 minutes.
2. Serial alcohol rehydration process (same as H&E staining)
3. Put it in a PBS washing tank 3 times (5min each)

Antigen retrieval (Choose one method)

Enzyme activation:

1. Incubate PBS containing 0.1% trypsin or 0.1% protease at 37℃ for 2 to 30 minutes
2. Rinse for 10 minutes on PBS.

Activation by microwave:

1. Prepare citric acid 500mL (50mL of citric acid pH 6.0 + 450 mL of MilliQ water)
2. Microwave citric acid at 600W until the bubble can be seen (around 5 min)
3. Put slides into citric acid and microwave at 200W for 5 min
4. Put slides into PBS and wait until cooldown (around 30 min)

Inactivation of endogenous peroxidase

1. Rinse slides 3 times with 1X TBS for 1 min each

2. Incubate slides in 3% H2O2 (diluted in distilled water) at room temperature for 10 min
3. Rinse slides 3 times with 1X TBS for 1 min each

Primary antibody reaction

1. Incubate slides in 3% BSA (diluted in TBS) at room temperature for 60 min

2. Incubate slides with Integrin beta4 antibody for 90 min at room temperature
3. Rinse slides 3 times with 1X TBS-T for 1 min each

Secondary antibody reaction

1. Incubate slides in 1:200 secondary antibody at 37C for 60 min

2. Rinse slides 3 times with 1X TBS-T for 1 min each
3. DAB staining (Refer to DAB protocols)