TITLE: Immunohistochemistry Protocol for Paraffin-Embedded Sections  - General
AUTHORS: Julianna Tomlinson, Nathalie Lengacher

[DESCRIPTION]
This protocol details general immunohistochemistry procedure.

[STEPS]
SECTION: Day 1 - Deparaffinize
1. Dip slides in 100% Citrisolv 3 x 5 mins.

SECTION: Hydration
2. Dip in 100% EtOH  2 x 2 mins.

SECTION: Hydration
4. Dip in 80% EtOH 2 x 2 mins.

SECTION: Hydration
5. Dip in ddH2O 2 x 2 mins.

SECTION: Quench endogenous peroxides
6. Incubate slides in 0.3% H2O2 in methanol for 15 min (500 µL of 30% H2O2 in 50 mL  MetOH, in white containers).

SECTION: Antigen retrieval
7. 
 


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

SECTION: Antigen retrieval
8. Transfer slides to white containers with AR solution. Microwave for 10 min at power level 1.

SECTION: Antigen retrieval
9. Rest slides at 4 Room temperature for 20 min, change water in bottom of container.

SECTION: Wash
10. Remove AR solution and wash slides with ddH2O for 5 min.

SECTION: Wash
11. Draw bubble with hydrophobic pen during this wash.
SECTION: Wash
12. Rinse slides with PBS for 5 min.

SECTION: Blocking
13. Block with 5% normal goat serum in PBS + 0.1% Triton-x-100 + 0.05% Tween-20, 30 min @ 4 Room temperature (300 µL /slide).

SECTION: Primary antibody
14. Dilute antibody in 5% normal goat serum in PBS + 0.1% Triton-x-100 + 0.05% Tween-20, 5 min at  4 °C(300 µL/slide).

SECTION: Day 2 - Wash
15. Rinse with PBS; 3 x 5 minutes
SECTION: Secondary antibody
16. Add secondary antibody diluted 1:225 in PBS + 0.1% triton-X + 0.05% tween-20 with 5% normal goat serum (~300 µL/slide).

SECTION: Secondary antibody
16.1. 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).
SECTION: Secondary antibody
17.  Incubate at Room temperature for 60 min  -  120 min.

SECTION: Wash
18. Rinse with PBS; 3 x 5 minutes.
SECTION: Substrate development
22. 
 


For regular DAB:
SECTION: Substrate development
22.1. Add 1 mL of DAB to 150 mL of 50 millimolar (mM) Tris +50 µL 30% H202.

SECTION: Substrate development
22.2. Dip slides into DAB solution, develop until colour appears ~ 3 min.

SECTION: Substrate development
23. For eDAB (SIGMAFAST™ DAB with Metal Enhancer):
SECTION: Substrate development
23.1. Add 1 tablet DAB/Cobalt and 1 tablet Urea Hydrogen Peroxide to 5 mL ddH2O, vortex until dissolved.


 

SECTION: Substrate development
23.2. Use ~300 µL/slide, develop until colour appears ~ 3 min.

SECTION: Substrate development
24. Rinse with PBS to stop reaction.
SECTION: Substrate development
25. Rinse with ddH20 for 5 min.

SECTION: Counterstain
26. Counterstain with 1:2 Hematoxylin:ddH2O, dip slides in quickly.
SECTION: Counterstain
27. Remove excess hematoxylin in ddH2O; 2X.
SECTION: Dehydration
28. 5 dips, 1 minute each:

 
SECTION: Dehydration
28.1. Dip in 50% EtOH 1 min

SECTION: Dehydration
28.2. Dip in 80% EtOH x2 1 min.

SECTION: Dehydration
28.3. Dip in 95% EtOH 1 min.

SECTION: Dehydration
28.4. Dip in 100% EtOH x2 1 min.

SECTION: Dehydration
28.5. Dip in 1:1 Citrisolv: 100% EtOH 1 min.

SECTION: Dehydration
28.6. Dip in 100% Citrisolv x2 1 min.

SECTION: Coverslip
29. Coverslip with permount solution, allow slides to dry before viewing on microscope.
SECTION: Amplification
19. Prepare amplification solution at least 30 min before use.

SECTION: Amplification
20. Amplification with VECTASTAIN® Elite® ABC HRP Kit.

SECTION: Amplification
20.1. 9 µL solution A per mL PBS, vortex; + 9 µL solution B per mL PBS, vortex; (300 µL/slide).

SECTION: Amplification
20.2. Incubate at Room temperature for 60 min-120 min.

SECTION: Wash
21. Rinse slides with PBS; 3 x 5 minutes
SECTION: Wash
21.1. Rinse slides with PBS; 3 x 5 min (1/3).

SECTION: Wash
21.3. Rinse slides with PBS; 3 x 5 min (3/3).
SECTION: Wash
21.2. Rinse slides with PBS; 3 x 5 min (2/3).
SECTION: Day 1 - Deparaffinize
1.1. Dip slides in 100% Citrisolv 3 x 5 min (1/3).
SECTION: Day 1 - Deparaffinize
1.3. Dip slides in 100% Citrisolv 3 x 5 min (3/3).
SECTION: Day 1 - Deparaffinize
1.2. Dip slides in 100% Citrisolv 3 x 5 min (2/3).
SECTION: Hydration
2.1. Dip in 100% EtOH 2 x 2 min (1/2).
SECTION: Hydration
2.2. Dip in 100% EtOH 2 x 2 min (2/2).
SECTION: Hydration
3. Dip in 95% EtOH 2 x 2 mins.
SECTION: Hydration
3.1. Dip in 95% EtOH 2 x 2 min (1/2).
SECTION: Hydration
3.2. Dip in 95% EtOH 2 x 2 min (2/2).
SECTION: Hydration
4.1. Dip in 80% EtOH 2 x 2 min (1/2).
SECTION: Hydration
4.2. Dip in 80% EtOH 2 x 2 min (2/2).
SECTION: Hydration
5.1. Dip in ddH2O 2 x 2 min (1/2).
SECTION: Hydration
5.2. Dip in ddH2O 2 x 2 min (2/2).
SECTION: Day 2 - Wash
15.1. Rinse with PBS; 3 x 5 min (1/3).
SECTION: Day 2 - Wash
15.3. Rinse with PBS; 3 x 5 min (3/3).
SECTION: Day 2 - Wash
15.2. Rinse with PBS; 3 x 5 min (2/3).
SECTION: Wash
18.1. Rinse slides with PBS; 3 x 5 min (1/3).
SECTION: Wash
18.3. Rinse slides with PBS; 3 x 5 min (3/3).
SECTION: Wash
18.2. Rinse slides with PBS; 3 x 5 min (2/3).