TITLE: DAB staining protocol for subsequent stereological cell counting
AUTHORS: Martin T. Henrich, Fanni F. Geibl

[DESCRIPTION]
This protocol describes the steps for performing a double chromogen staining using DAB and SK- 4700. Stained sections can subsequently be imaged and used for stereological cell quantification with Stereo Investigator software from MBF Bioscience or other Stereology software.

[STEPS]
SECTION: DAB staining – Before the procedure:
1. Prepare 0.1 Molarity (M) PB (PB).

SECTION: DAB Staining Procedure
5. Place brain sections ( - ) in 12-well-plates with netwell inserts (up to 6-10 sections per netwell insert, depending on the size of the sections).
SECTION: DAB Staining Procedure
7. Quench sections for 15 min at Room temperature in quenching solution in 12-well-plates on plajorm shaker.
 


SECTION: DAB Staining Procedure
8. Thereafter, wash sections for 4x 5 min in 0.1 Molarity (M) PB on platform shaker. Exchange PB solution in between washing steps.

SECTION: DAB Staining Procedure
9. Block sections for 60 min at Room temperature in NDS solution in 12-well-plates.
 


SECTION: DAB Staining Procedure
10. For incubation with primary antibody (e.g. anti neuronal nuclei (NeuN), Merck Millipore, MAB377, 1:1000), transfer sections in a new 12-well-plate but without netwell inserts. This allows better shaking 60 min. Solution for incubation with primary antibody should contain NDS solution and the respective primary antibody diluted according to manufacturer recommendation. Incubate sections on orbital shaker with gentle shaking at 4 °C 60 min.
 

SECTION: DAB staining – Before the procedure:
2. Prepare PBT (0.1 Molarity (M) PB with 0.3% Triton X-100).

SECTION: DAB Staining Procedure
6. Place 12-well-plates on platform shaker and wash sections for 3x 5 min in 0.1 Molarity (M) PB. Exchange PB solution in between washing steps.

SECTION: DAB staining – Before the procedure:
3. Prepare a quenching solution (for 40 mL solution: 32 mL 0.1 Molarity (M) PB, 4 mL methanol (100%), 4 mL H2O2 (30%)).

SECTION: DAB staining – Before the procedure:
4. Prepare NDS solution (5% normal donkey serum diluted in PBT).
SECTION: DAB Staining Procedure
6.1. Place 12-well-plates on platform shaker and wash sections for 5 min in 0.1 Molarity (M) PB. Exchange PB solution in between washing steps (1/3).

SECTION: DAB Staining Procedure
6.3. Place 12-well-plates on platform shaker and wash sections for 5 min in 0.1 Molarity (M) PB. Exchange PB solution in between washing steps (3/3).
SECTION: DAB Staining Procedure
6.2. Place 12-well-plates on platform shaker and wash sections for 5 min in 0.1 Molarity (M) PB. Exchange PB solution in between washing steps (2/3).
SECTION: DAB Staining Procedure
8.1. Thereafter, wash sections for 5 min in 0.1 Molarity (M) PB on plajorm shaker. Exchange PB solution in between washing steps (1/).

SECTION: DAB Staining Procedure
8.3. Thereafter, wash sections for 5 min in 0.1 Molarity (M) PB on plajorm shaker. Exchange PB solution in between washing steps (3/4).
SECTION: DAB Staining Procedure
8.2. Thereafter, wash sections for 5 min in 0.1 Molarity (M) PB on plajorm shaker. Exchange PB solution in between washing steps (2/4).
SECTION: DAB Staining Procedure
8.4. Thereafter, wash sections for 5 min in 0.1 Molarity (M) PB on plajorm shaker. Exchange PB solution in between washing steps (4/4).
SECTION: DAB Staining Procedure
11. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections 4x 5 min in PB on a platform shaker at Room temperature. Change washing solution after each washing step.

SECTION: DAB Staining Procedure
11.1. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections for 5 min in PB on platform shaker at Room temperature. Change washing solution after each washing step (1/4).
SECTION: DAB Staining Procedure
11.2. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections for 5 min in PB on platform shaker at Room temperature. Change washing solution after each washing step (2/4).
SECTION: DAB Staining Procedure
11.4. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections for 5 min in PB on platform shaker at Room temperature. Change washing solution after each washing step (4/4).
SECTION: DAB Staining Procedure
11.3. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections for 5 min in PB on platform shaker at Room temperature. Change washing solution after each washing step (3/4).
SECTION: DAB Staining Procedure
12. Next, transfer sections back in 12-well-plate without netwell inserts for incubation with biotinylated secondary antibody (e.g., biotinylated donkey anti-mouse, Jackson ImmunoResearch, 715-065-151, 1:500) diluted in NDS solution. Incubate for 60 min on orbital shaker at Room temperature.
 


SECTION: DAB Staining Procedure
13. Next, without washing, incubate sections in 12-well-plate without netwell inserts on orbital shaker at Room temperature for 60 min in avidin-biotin-peroxidase solution using the Vectastain ABC Kit, diluted according to manufacturer’s recommendation.

SECTION: DAB Staining Procedure
14. Prepare 5% DAB working solution fresh before color reaction (for 40 ml solution: 35.2 mL 0.1 Molarity (M) PB, 4 mL DAB solution (5 1629), 0.8 mL H2O2 (1%)). Wear an FFP2/N95 mask or a respirator for this step and all further steps in which you work directly with DAB. Only carry out this work under a fume hood.

SECTION: DAB Staining Procedure
15. Without washing, transfer brain sections back in 12-well-plates with netwell inserts and start DAB color reaction by incubating sections in DAB working solution for 3-6 min at Room temperature under fume hood, depending on desired color intensity. Gently shake sections during incubation to avoid sections sticking together.

SECTION: DAB Staining Procedure
16. Stop color reaction by washing sections 4x 5 min. in 0.1 Molarity (M) PB on plajorm shaker. Exchange PB solution in between washing steps. Use a fresh 12-well-plate which was not previously in contact with DAB solution. While washing use diluted Na-hypochlorite solution to neutralize DAB solution on used 12-well-plate and inserts. Let plastics neutralize in diluted Nahypochlorite solution 5 min.

SECTION: DAB Staining Procedure
17. Block sections again for 60 min at Room temperature in NDS solution in 12-well-plates on orbital shaker.
 


SECTION: DAB Staining Procedure
18. For incubation with primary antibody (e.g. anti-tyrosine hydroxylase (TH), Merck Millipore, AB152, 1:1000), transfer sections in a new 12-well-plate but without netwell inserts. This allows better shaking 60 min. solution for incubation with primary antibody should contain NDS solution and the respective primary antibody diluted according to manufacturer recommendation. Incubate sections on orbital shaker with gentle shaking at 4 °C 60 min.
 

SECTION: DAB Staining Procedure
19. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections 4x 5 min in PB on platform shaker at Room temperature. Change washing solution after each washing step.

SECTION: DAB Staining Procedure
20. Next, transfer sections back in 12-well-plate without netwell inserts for incubation with biotinylated secondary antibody (e.g., biotinylated donkey anti-rabbit, Jackson ImmunoResearch, 711-065-152, 1:500) diluted in NDS solution. Incubate for 60 min on orbital shaker at Room temperature.
 


SECTION: DAB Staining Procedure
21. Next, without washing, incubate sections in 12-well-plate without netwell inserts on orbital shaker at Room temperature for 60 min in avidin-biotin-peroxidase solution using the Vectastain ABC Kit, diluted according to manufacturer’s recommendation.

SECTION: DAB Staining Procedure
22. Prepare Peroxidase Substrate Kit (SG Peroxidase Substrate Kit, Vector Labs, SK-4700) as recommended by the manufacturer. Wear an FFP2/N95 mask or a respirator for this step and all further steps in which you work directly with SK-4700. Only carry out this work under a fume hood.
SECTION: DAB Staining Procedure
23. Without washing, transfer brain sections back in 12-well-plates with netwell inserts and start SK-4700 color reaction by incubating sections in prepared peroxidase substrate solution for 3- 6 min at Room temperature under fume hood, depending on desired color intensity. Gently shake sections during incubation to avoid sections sticking together.

SECTION: DAB Staining Procedure
24. Stop color reaction by washing sections 4x 5 min. in 0.1 Molarity (M) PB on platform shaker. Exchange PB solution in between washing steps. Use a fresh 12-well-plate which was not previously in contact with SK-4700 solution. While washing use diluted Na-hypochlorite solution to neutralize SK-4700 solution on used 12-well-plate and inserts. Let plastics neutralize in diluted Na-hypochlorite solution 5 min.

SECTION: DAB Staining Procedure
25. After washing, mount sections on microscopy slides using a fine brush.
SECTION: DAB Staining Procedure
26. Let sections dry 5 min.

SECTION: DAB Staining Procedure
27. Immerse dried slides in a series of ethanol (70%, 96%, 100%) 30 sec. each for stepwise dehydration. Work under a fume hood.
SECTION: DAB Staining Procedure
28. Let slides rest for 10 min in 100% xylene solution. Work under a fume hood.

SECTION: DAB Staining Procedure
29. Next, quickly apply a small amount of hard-drying mounting medium (e.g., EukiS) sufficient to cover the sections. Carefully avoid the formation of air bubbles. Gently apply a coverslip over the sections and the mounting medium. Work under a fume hood.
SECTION: DAB Staining Procedure
16.1. Stop color reaction by washing sections for 5 min in 0.1 Molarity (M) PB on plajorm shaker (1/4).
SECTION: DAB Staining Procedure
16.4. Stop color reaction by washing sections for 5 min in 0.1 Molarity (M) PB on plajorm shaker (4/4).

SECTION: DAB Staining Procedure
16.3. Stop color reaction by washing sections for 5 min in 0.1 Molarity (M) PB on plajorm shaker (3/4).

SECTION: DAB Staining Procedure
16.2. Stop color reaction by washing sections for 5 min in 0.1 Molarity (M) PB on plajorm shaker (2/4).
SECTION: DAB Staining Procedure
19.1. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections for 5 min in PB on platform shaker at Room temperature. Change washing solution after each washing step (1/4).

SECTION: DAB Staining Procedure
19.4. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections for 5 min in PB on platform shaker at Room temperature. Change washing solution after each washing step (4/4).
SECTION: DAB Staining Procedure
19.2. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections for 5 min in PB on platform shaker at Room temperature. Change washing solution after each washing step (2/4).

SECTION: DAB Staining Procedure
19.3. On the next day, transfer sections into 12-well-plate with netwell inserts and wash sections for 5 min in PB on platform shaker at Room temperature. Change washing solution after each washing step (3/4).

SECTION: DAB Staining Procedure
24.1. Stop color reaction by washing sections for 5 min in 0.1 Molarity (M) PB on platform shaker (1/4).

SECTION: DAB Staining Procedure
24.2. Stop color reaction by washing sections for 5 min in 0.1 Molarity (M) PB on platform shaker (2/4).
SECTION: DAB Staining Procedure
24.4. Stop color reaction by washing sections for 5 min in 0.1 Molarity (M) PB on platform shaker (4/4).
SECTION: DAB Staining Procedure
24.3. Stop color reaction by washing sections for 5 min in 0.1 Molarity (M) PB on platform shaker (3/4).
SECTION: DAB staining – After the procedure:
30. Let slides cure for 2 days under a fume hood.
SECTION: DAB staining – After the procedure:
31. Dispose of waste and excess reagents/solution according to institutional guidelines.
SECTION: DAB staining – After the procedure:
32. Clean tools/working station.
SECTION: DAB staining – After the procedure:
33. Microscopy slides should be stored in a designated container at room temperature until time of observation.
SECTION: Stereological cell counting – Before the procedure:
34. Turn on microscope and computer according to specific manuals/instructions.
SECTION: Stereological cell counting – Before the procedure:
35. Turn on MBF Stereo Investigator software.
SECTION: Stereological cell counting – Procedure:
36. Load microscopy slides in the designated stage.
SECTION: Stereological cell counting – Procedure:
37. Use brightfield settings for imaging.
SECTION: Stereological cell counting – Procedure:
38. Carefully follow all steps of the Stereo Investigator Workflow according to the manual. The workflow is step-by-step and very intuitive.
SECTION: Stereological cell counting – Procedure:
39. We typically use a 60x oil immersion lens for imaging.
SECTION: Stereological cell counting – Procedure:
40. After cell counts have been collected export counting data into an excel sheet for further data analysis.
SECTION: Stereological cell counting – After the procedure:
41. Clean immersion objectives with a lens wipe and the appropriate cleaning solution.
SECTION: Stereological cell counting – After the procedure:
42. Turn off software/microscope/laser according to specific instructions.