TITLE: DAB ANTIBODY (IHC) STAINING PROTOCOL
AUTHORS: Michael Lee

[DESCRIPTION]
This is the basic protocol for antibody staining of formalin fixed paraffin embedded (FFPE) tissue.

[GUIDELINES]
Principle:

For antibody staining to be successful, most FFPE tissue requires antigen retrieval of some kind. Formalin fixation cross-links proteins during the course of fixation. Antigen retrieval unlinks the proteins and opens up the antigen sites so that the antibody will be able to bind to them.

References:
Carson, Freida, Histotechnology – A Self-Instructional Text, 2nd Edition, ASCP Press, 1997

[STEPS]
SECTION: Deparaffinize tissue: Day 1
1. 2 options: 

1) Either place slides on a slide warmer with temperature set to approx. 57 °C. Leave the slides on the warming plate until the paraffin looks melted on all of the slides (about 10 - 15 min) or 2)  Put slides in a 60 °C oven for about 30 min.

SECTION: Deparaffinize tissue: Day 1
2. Place slides vertically in the gray plastic slide holders and run the slides through the following solutions in the staining set up located in the fume hood.

 

3. Fill the vegetable steamer with deionized water to the second line in the transparent corner section of the steamer.  
4. Add capillary gap slides (same number as the number of slides you are staining) in the slide holder to wet the slides.  
4.1. Fill the plastic “boat” containers with approx. 20 mL of one of the antigen retrieval solutions (Reveal or citrate buffer).

4.2. Pick up a set of slides (rough side of the capillary gap slide facing the tissue section slide) and put the slides into one of the troughs in the boat container.
4.3. Push slides toward each other and allow the fluid to come up slowly between the two slides.
 

4.4. Steam for about 30 min.

3.1. Turn steamer on and push the up arrow button until the time is about 40 min.
 

5. Remove clear basket from the steamer and allow slides to cool for about 20 min to Room temperature.

6. Transfer the “boats” of slides to the inner steamer container and rinse with running water for about 20 min. 

6.1. Unpeel the slides from each other and transfer the slides to the gray slide holder for a final rinse (~5 min).

SECTION: Deparaffinize tissue: Day 1
3. 
 

Microwave slides/solution for 5 min. on high power (~700 watts). 

SECTION: Deparaffinize tissue: Day 1
3.1. Make sure slides are still covered with retrieval solution or add fresh solution and repeat microwaving.
SECTION: Deparaffinize tissue: Day 1
3.2. This process can be repeated 2-3 times.
SECTION: Deparaffinize tissue: Day 1
3.3. Let slides slowly cool in the coplin jar to Room temperature for approx.20 min.

SECTION: Staining steps:
7. 
Dilute or use prediluted TRIS (1x concentration) for 10 min, approx 250- 300 µL per slide.
 
SECTION: Staining steps:
8. 100% Background Sniper for nearly 13 min. Do not go longer than 15 min. (alternative: 10% normal goat serum made in 1X TRIS or PBS can be substituted for 60 min).

SECTION: Staining steps:
9. Make up antibody solution at desired concentration in a diluent of5% Sniper in 1X TRIS solution. Using approx. 100 µL per slide, make up enough antibody to cover all slides.

SECTION: Staining steps:
10. Cover the slides with either a glass coverslip (24 x 60 mm) or parafilm and put in a 4 °C. refrigerator 60 min.

SECTION: Staining steps:
4. Dilute or use prediluted TRIS (1x concentration) for 10 min, approx 250 µL- 300 µL per slide.
 
SECTION: Staining steps:
5. 100% Background Sniper for nearly 13 min. Do not go longer than 15 min. (alternative: 10% normal goat serum made in 1X TRIS or PBS can be substituted for 60 min).

SECTION: Staining steps:
6. Make up antibody solution at desired concentration in a diluent of5% Sniper in 1X TRIS solution. Using approx. 100 µL per slide, make up enough antibody to cover all slides.
SECTION: Staining steps:
7. Cover the slides with either a glass coverslip (24 x 60 mm) or parafilm and put in a 4 °C. refrigerator 60 min.
SECTION: DAY 2:
11. Take slides out of the refrigerator and warm up to Room temperature about 15 min- 20 min. Remove the cover slips and rinse the slides with 1X TRIS solution, then incubate slides in 1X TRIS solution for about 5 min x 2.

 
SECTION: DAY 2:
12. Secondary reagent/antibody for 30 min
Apply the secondary reagent appropriate to the antibody, i.e., if your antibody was raised in a rabbit, you will apply an anti- Rabbit link.

 


SECTION: DAY 2:
13. Rinse slides with 1X TRIS, 10 min.

SECTION: DAY 2:
14. 3% Hydrogen Peroxide made in 1X TRIS for 10 min x 2.Rinse between with 1X TRIS.

SECTION: DAY 2:
15. Rinse slides with 1X TRIS, 5 minutes x 2.

SECTION: DAY 2:
8. Take slides out of the refrigerator and warm up to Room temperature about 15 min- 20 min. Remove the cover slips and rinse the slides with 1X TRIS solution, then incubate slides in 1X TRIS solution for about 5 min x 2.

SECTION: DAY 2:
9. Secondary reagent/antibody for 30 min.
Apply the secondary reagent appropriate to the antibody, i.e., if your antibody was raised in a rabbit, you will apply an anti- Rabbit link.
 

SECTION: DAY 2:
10. Rinse slides with 1X TRIS, 10 min.
SECTION: DAY 2:
11. 3% Hydrogen Peroxide made in 1X TRIS for 10 min x 2. Rinse between with 1X TRIS.
SECTION: DAY 2:
12. Rinse slides with 1X TRIS, 5 minutes x 2.
SECTION: DAY 2:
16. TERTIARY Reagent for 30 min.
 
SECTION: DAY 2:
17. Rinse slides with 1X TRIS, 5 minutes x 2.


SECTION: DAY 2:
16.1. The Tertiary Reagent is made up about 25 min before you apply it.
 



SECTION: DAY 2:
18. DAB (DIAMINOBENZINE) chromogen for approximately 3 min to develop.
 


SECTION: DAY 2:
18.1. Make up enough DAB solution to cover all the slides, using 250 µL- 300 µL per slide as a guide.
 



SECTION: DAY 2:
18.2. Filter the DAB solution with a 0.22 µL syringe filter into a new vial before use.
 



SECTION: DAY 2:
18.3. After development put slides in slide holder in water container.
SECTION: DAY 2:
18.4. Fill clear basket with water then submerge slides in running water in sink.
SECTION: DAY 2:
18.5. Now counterstain.
SECTION: DAY 2:
13. DAB (DIAMINOBENZINE) chromogen for approximately 3 min to develop.
 


SECTION: DAY 2:
13.1. Make up enough DAB solution to cover all the slides, using 250 µL- 300 µL per slide as a guide.
 



SECTION: DAY 2:
13.2. Filter the DAB solution with a 0.22 µL syringe filter into a new vial before use.
 


SECTION: DAY 2:
13.3. After development put slides in slide holder in water container.
SECTION: DAY 2:
13.4. Fill clear basket with water then submerge slides in running water in sink.
SECTION: DAY 2:
13.5. Now counterstain.
SECTION: Counterstaining with hematoxylin and coverslipping:
19. 
Filtered Harris Hematoxylin – several dips, return to water and check after a few minutes to see if the hematoxylin is strong enough, if not, return for more dips and repeat.

SECTION: Counterstaining with hematoxylin and coverslipping:
20. Tap water rinse for about 5 min to both “blue” the hematoxylin and rinse the slides until the water runs clear. Put slides in container with filtered water for dehydration and clearing steps as follows:

 

 


Result examples:

Nuclei = Blue
Positive staining antigen = Brown
Background & cytoplasm= pale blue to colorless

  
 
SECTION: Counterstaining with hematoxylin and coverslipping:
14. Filtered Harris Hematoxylin – several dips, return to water and check after a few minutes to see if the hematoxylin is strong enough, if not, return for more dips and repeat.

SECTION: Counterstaining with hematoxylin and coverslipping:
15. Tap water rinse for about 5 min to both “blue” the hematoxylin and rinse the slides until the water runs clear. Put slides in container with filtered water for dehydration and clearing steps as follows:

 

 

 



Result examples:


Nuclei = Blue
Positive staining antigen = Brown
Background & cytoplasm= pale blue to colorless


 

SECTION: DAY 2:
15.1. Rinse slides with 1X TRIS, 5 min x 2 (1/2).
SECTION: DAY 2:
15.2. Rinse slides with 1X TRIS, 5 min x 2 (2/2).
SECTION: DAY 2:
17.1. Rinse slides with 1X TRIS, 5 min x 2 (1/2).
SECTION: DAY 2:
17.2. Rinse slides with 1X TRIS, 5 min x 2 (2/2).
SECTION: DAY 2:
12.1. Rinse slides with 1X TRIS, 5 min x 2 (1/2).
SECTION: DAY 2:
12.2. Rinse slides with 1X TRIS, 5 min x 2 (2/2)