Immunohistochemistry Protocol for Paraffin-Embedded Sections - General

Dip slides in 100% Citrisolv 3 x 5 mins.

Dip slides in 100% Citrisolv 3 x 0h 5m 0s (1/3).

Dip slides in 100% Citrisolv 3 x 0h 5m 0s (2/3).

Dip slides in 100% Citrisolv 3 x 0h 5m 0s (3/3).

Dip in 100% EtOH 2 x 2 mins.

Dip in 100% EtOH 2 x 0h 2m 0s (1/2).

Dip in 100% EtOH 2 x 0h 2m 0s (2/2).

Dip in 95% EtOH 2 x 2 mins.

Dip in 95% EtOH 2 x 0h 2m 0s (1/2).

Dip in 95% EtOH 2 x 0h 2m 0s (2/2).

Dip in 80% EtOH 2 x 2 mins.

Dip in 80% EtOH 2 x 0h 2m 0s (1/2).

Dip in 80% EtOH 2 x 0h 2m 0s (2/2).

Dip in ddH2O 2 x 2 mins.

Dip in ddH₂O 2 x 0h 2m 0s (1/2).

Dip in ddH₂O 2 x 0h 2m 0s (2/2).

Incubate slides in 0.3% H₂O₂ in methanol for 0h 15m 0s (500µL of 30% H₂O₂ in 50mL MetOH, in white containers).

Pre-heat antigen retrieval solution in microwave in white containers (~50mL), for 0h 5m 0s at full power. Use large plastic container and fill halfway with water.

Transfer slides to white containers with AR solution. Microwave for 0h 10m 0s at power level 1.

Rest slides at 4Room temperature for 0h 20m 0s, change water in bottom of container.

Remove AR solution and wash slides with ddH₂O for 0h 5m 0s.

Draw bubble with hydrophobic pen during this wash.

Rinse slides with PBS for 0h 5m 0s.

Block with 5% normal goat serum in PBS + 0.1% Triton-x-100 + 0.05% Tween-20, 0h 30m 0s @ 4Room temperature (300µL /slide).

Dilute antibody in 5% normal goat serum in PBS + 0.1% Triton-x-100 + 0.05% Tween-20, 0h 5m 0s at 4°C(300µL/slide).

Rinse with PBS; 3 x 5 minutes

Rinse with PBS; 3 x 0h 5m 0s (1/3).

Rinse with PBS; 3 x 0h 5m 0s (2/3).

Rinse with PBS; 3 x 0h 5m 0s (3/3).

Add secondary antibody diluted 1:225 in PBS + 0.1% triton-X + 0.05% tween-20 with 5% normal goat serum (~300µL/slide).

Commonly: biotinylated anti-rabbit IgG (H+L) made in goat (Vector Labs,BA-1000) or biotinylated anti mouse IgG (H+L) made in goat (Vector Labs, BA-9200).

Incubate at Room temperature for 1h 0m 0s - 2h 0m 0s.

Rinse with PBS; 3 x 5 minutes.

Rinse slides with PBS; 3 x 0h 5m 0s (1/3).

Rinse slides with PBS; 3 x 0h 5m 0s (2/3).

Rinse slides with PBS; 3 x 0h 5m 0s (3/3).

Prepare amplification solution at least 0h 30m 0s before use.

Amplification with VECTASTAIN® Elite® ABC HRP Kit.

9µL solution A per mL PBS, vortex; + 9µL solution B per mL PBS, vortex; (300µL/slide).

Incubate at Room temperature for 1h 0m 0s-2h 0m 0s.

Rinse slides with PBS; 3 x 5 minutes

Rinse slides with PBS; 3 x 0h 5m 0s (1/3).

Rinse slides with PBS; 3 x 0h 5m 0s (2/3).

Rinse slides with PBS; 3 x 0h 5m 0s (3/3).

For regular DAB:

Add 1mL of DAB to 150mL of 50millimolar (mM) Tris +50µL 30% H₂0₂.

Dip slides into DAB solution, develop until colour appears ~ 0h 3m 0s.

For eDAB (SIGMAFAST™ DAB with Metal Enhancer):

Add 1 tablet DAB/Cobalt and 1 tablet Urea Hydrogen Peroxide to 5mL ddH₂O, vortex until dissolved.

Use ~300µL/slide, develop until colour appears ~ 0h 3m 0s.

Rinse with PBS to stop reaction.

Rinse with ddH₂0 for 0h 5m 0s.

Counterstain with 1:2 Hematoxylin:ddH₂O, dip slides in quickly.

Remove excess hematoxylin in ddH₂O; 2X.

5 dips, 1 minute each:

Dip in 50% EtOH 0h 1m 0s

Dip in 80% EtOH x2 0h 1m 0s.

Dip in 95% EtOH 0h 1m 0s.

Dip in 100% EtOH x2 0h 1m 0s.

Dip in 1:1 Citrisolv: 100% EtOH 0h 1m 0s.

Dip in 100% Citrisolv x2 0h 1m 0s.

Coverslip with permount solution, allow slides to dry before viewing on microscope.