TITLE: DAB immunostaining of thin, fixed mouse brain tissue sections using HNA or NCAM to characterize human iPSC-derived cell xenografts
AUTHORS: Benjamin Trist, Ashish Mathai, Asheeta Prasad

[DESCRIPTION]
This protocol describes our use of chromogenic 3,3′-diaminobenzidine (DAB) immunohistochemistry to identify human iPSC-derived cells within thin, fixed mouse brain tissue section series’. We apply this workflow for post-mortem assessment of the survival and growth of human iPSC-derived cells which have been transplanted into the living brain of athymic mice.

[STEPS]
SECTION: Day 1 (~4-6 hrs)
1. Pre-heat oven and AR buffer to 70 °C.
SECTION: Day 1 (~4-6 hrs)
2. Label scintillation vials to match labels on section storage plates (mouse and section series IDs, name, date etc.).
SECTION: Day 1 (~4-6 hrs)
3. Transfer sections into scintillation vials using a transfer pipette or fine paintbrush.
SECTION: Day 1 (~4-6 hrs)
4. Remove anti-freeze solution and perform 3x 7 min washes in 1x PBS at Room temperature with gentle agitation.
Slow shaking on an orbital rocker recommended for washes/incubations to ensure even contact with solutions.
Use a glass pipette and rubber teat to remove solution during wash changes.
Anti-freeze solution must be rinsed off prior to immunostaining.
SECTION: Antigen retrieval (AR)
5. Incubate sections in AR buffer for 30 min at 70 °C.
SECTION: Antigen retrieval (AR)
6. Preheat AR buffer to 70 °C prior to addition to sections.
SECTION: Antigen retrieval (AR)
7. After antigen retrieval, allow sections to cool for 30 min before proceeding with staining.
SECTION: Antigen retrieval (AR)
8. Perform 3x 7 min washes in 1x PBST with agitation.
SECTION: Quenching step
9. Incubate sections in quenching solution for 30 min at Room temperature with gentle agitation.
SECTION: Quenching step
10. Perform 3x 7 min washes in 1x PBST with gentle agitation.
SECTION: Blocking step
11. Incubate sections in blocking solution for 60 min at Room temperature with gentle agitation.
SECTION: Primary antibody step
12. Incubate sections with NCAM (1:20,000) or HNA (1:15,000) primary antibodies diluted in blocking buffer   at 4 °C with gentle agitation.

SECTION: Day 2 (~8hrs)
13. Perform 3x 7 min washes in 1x PBST with gentle agitation.
SECTION: Secondary antibody step
14. Incubate sections in anti-rabbit biotinylated secondary antibody 1:500 diluted in blocking buffer that has been diluted 2-fold for 120 min at Room temperature.
SECTION: Secondary antibody step
15. Perform 3x 7 min washes in 1x PBST with gentle agitation.
SECTION: Tertiary complex step
16. Incubate sections in Avidin-Biotin Complex (ABC) kit solution (Vector Laboratories) for 120 min at Room temperature.
SECTION: Tertiary complex step
17. Prepare tertiary complex 30 min prior to use according to the manufacturer’s instructions.
100 µL A + 100 µL B + 9800 µL 1x PBS (1:100 A + 1:100 B in 1x PBS).
SECTION: Tertiary complex step
18. Perform 3x 7 min washes in 1x PBST with gentle agitation.
SECTION: Chromogen step
19. Perform DAB staining as follows;
SECTION: Chromogen step
20. Prepare DAB solution by dissolving 10 mg DAB tablet into 20 mL PBS (0.5 mg/mL).
SECTION: Chromogen step
21. Filter through Whatman paper #1 or 0.22 um syringe filter before use.
 

SECTION: Chromogen step
22. Prepare DAB-H2O2 solution immediately prior to use by adding 10 µL 30% H2O2 per 5 mL DAB solution and mix thoroughly.
SECTION: Chromogen step
23. Incubate sections with DAB-H2O2 solution for 8 min.
SECTION: Chromogen step
24. Perform 3x 7 min washes in 1x PBST with gentle agitation.

SECTION: Chromogen step
25. Mount tissue sections onto super-frost slides pre-coated with gelatin-chrome alum and allow to dry at Room temperature  .

SECTION: Day 3 (2 days later)
26. Process slide-mounted tissue sections through the following solutions; 
SECTION: Day 3 (2 days later)
27. dH2O 3 min.
SECTION: Day 3 (2 days later)
28. 50% ethanol 3 min.
SECTION: Day 3 (2 days later)
29. 70% ethanol 3 min .
SECTION: Day 3 (2 days later)
30. 95% ethanol 3 min .
SECTION: Day 3 (2 days later)
31. 100% ethanol 3 min.
SECTION: Day 3 (2 days later)
32. 100% ethanol 3 min .
SECTION: Day 3 (2 days later)
33. Xylene 5 min.
SECTION: Day 3 (2 days later)
34. Xylene 5 min.
SECTION: Day 3 (2 days later)
35. Xylene 5 min.
SECTION: Day 3 (2 days later)
36. Coverslip slides with DEPEX mounting media and allow to dry in the fume hood   before proceeding with microscopy.

SECTION: Day 3 (2 days later)
37. Image sections using bright field microscopy for subsequent xenograft characterization.
SECTION: Day 1 (~4-6 hrs)
4.1. Remove anti-freeze solution and wash in 1x PBS at Room temperature for 7 min(1/3).
SECTION: Day 1 (~4-6 hrs)
4.2. Remove anti-freeze solution and wash in 1x PBS at Room temperature for 7 min(2/3).
SECTION: Day 1 (~4-6 hrs)
4.3. Remove anti-freeze solution and wash in 1x PBS at Room temperature for 7 min(3/3).
SECTION: Antigen retrieval (AR)
8.1. Wash in 1x PBST with agitation for 7 min(1/3).
SECTION: Antigen retrieval (AR)
8.3. Wash in 1x PBST with agitation for 7 min(3/3).
SECTION: Antigen retrieval (AR)
8.2. Wash in 1x PBST with agitation for 7 min(2/3).
SECTION: Quenching step
10.1. Wash in 1x PBST with gentle agitation for 7 min(1/3).
SECTION: Quenching step
10.2. Wash in 1x PBST with gentle agitation for 7 min(2/3).
SECTION: Quenching step
10.3. Wash in 1x PBST with gentle agitation for 7 min(3/3).
SECTION: Day 2 (~8hrs)
13.1. Wash in 1x PBST with gentle agitation for 7 min(1/3).
SECTION: Day 2 (~8hrs)
13.3. Wash in 1x PBST with gentle agitation for 7 min(3/3).
SECTION: Day 2 (~8hrs)
13.2. Wash in 1x PBST with gentle agitation for 7 min(2/3).
SECTION: Secondary antibody step
15.1. Wash in 1x PBST with gentle agitation for 7 min(1/3).
SECTION: Secondary antibody step
15.3. Wash in 1x PBST with gentle agitation for 7 min(3/3).
SECTION: Secondary antibody step
15.2. Wash in 1x PBST with gentle agitation for 7 min(2/3).
SECTION: Tertiary complex step
18.1. Wash in 1x PBST with gentle agitation for 7 min(1/3).
SECTION: Tertiary complex step
18.2. Wash in 1x PBST with gentle agitation for 7 min(2/3).
SECTION: Tertiary complex step
18.3. Wash in 1x PBST with gentle agitation for 7 min(3/3).
SECTION: Chromogen step
24.1. Wash in 1x PBST with gentle agitation for 7 min(1/3).
SECTION: Chromogen step
24.3. Wash in 1x PBST with gentle agitation for 7 min(3/3).
SECTION: Chromogen step
24.2. Wash in 1x PBST with gentle agitation for 7 min(2/3).