DAB immunostaining of thin, fixed mouse brain tissue sections using HNA or NCAM to characterize human iPSC-derived cell xenografts
Benjamin Trist, Asheeta Prasad, Ashish Mathai
Abstract
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.
Attachments
Steps
Day 1 (~4-6 hrs)
Pre-heat oven and AR buffer to 70°C.
Label scintillation vials to match labels on section storage plates (mouse and section series IDs, name, date etc.).
Transfer sections into scintillation vials using a transfer pipette or fine paintbrush.
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.
Remove anti-freeze solution and wash in 1x PBS at Room temperature for 0h 7m 0s(1/3).
Remove anti-freeze solution and wash in 1x PBS at Room temperature for 0h 7m 0s(2/3).
Remove anti-freeze solution and wash in 1x PBS at Room temperature for 0h 7m 0s(3/3).
Antigen retrieval (AR)
Incubate sections in AR buffer for 0h 30m 0s at 70°C.
Preheat AR buffer to 70°C prior to addition to sections.
After antigen retrieval, allow sections to cool for 0h 30m 0s before proceeding with staining.
Perform 3x 7 min washes in 1x PBST with agitation.
Wash in 1x PBST with agitation for 0h 7m 0s(1/3).
Wash in 1x PBST with agitation for 0h 7m 0s(2/3).
Wash in 1x PBST with agitation for 0h 7m 0s(3/3).
Quenching step
Incubate sections in quenching solution for 0h 30m 0s at Room temperature with gentle agitation.
Perform 3x 7 min washes in 1x PBST with gentle agitation.
Wash in 1x PBST with gentle agitation for 0h 7m 0s(1/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(2/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(3/3).
Blocking step
Incubate sections in blocking solution for 1h 0m 0s at Room temperature with gentle agitation.
Primary antibody step
Incubate sections with NCAM (1:20,000) or HNA (1:15,000) primary antibodies diluted in blocking buffer at 4°C with gentle agitation.
Day 2 (~8hrs)
Perform 3x 7 min washes in 1x PBST with gentle agitation.
Wash in 1x PBST with gentle agitation for 0h 7m 0s(1/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(2/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(3/3).
Secondary antibody step
Incubate sections in anti-rabbit biotinylated secondary antibody 1:500 diluted in blocking buffer that has been diluted 2-fold for 2h 0m 0s at Room temperature.
Perform 3x 7 min washes in 1x PBST with gentle agitation.
Wash in 1x PBST with gentle agitation for 0h 7m 0s(1/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(2/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(3/3).
Tertiary complex step
Incubate sections in Avidin-Biotin Complex (ABC) kit solution (Vector Laboratories) for 2h 0m 0s at Room temperature.
Prepare tertiary complex 0h 30m 0s prior to use according to the manufacturer’s instructions.
100µLA +100µLB +9800µL1x PBS (1:100 A + 1:100 B in 1x PBS).
Perform 3x 7 min washes in 1x PBST with gentle agitation.
Wash in 1x PBST with gentle agitation for 0h 7m 0s(1/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(2/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(3/3).
Chromogen step
Perform DAB staining as follows;
Prepare DAB solution by dissolving 10mg DAB tablet into 20mL PBS (0.5mg/mL).
Filter through Whatman paper #1 or 0.22 um syringe filter before use.
Prepare DAB-H2O2 solution immediately prior to use by adding 10µL 30% H2O2 per 5mL DAB solution and mix thoroughly.
Incubate sections with DAB-H2O2 solution for 0h 8m 0s.
Perform 3x 7 min washes in 1x PBST with gentle agitation.
Wash in 1x PBST with gentle agitation for 0h 7m 0s(1/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(2/3).
Wash in 1x PBST with gentle agitation for 0h 7m 0s(3/3).
Mount tissue sections onto super-frost slides pre-coated with gelatin-chrome alum and allow to dry at Room temperature .
Day 3 (2 days later)
Process slide-mounted tissue sections through the following solutions;
dH2O 0h 3m 0s.
50% ethanol 0h 3m 0s.
70% ethanol 0h 3m 0s .
95% ethanol 0h 3m 0s .
100% ethanol 0h 3m 0s.
100% ethanol 0h 3m 0s .
Xylene 0h 5m 0s.
Xylene 0h 5m 0s.
Xylene 0h 5m 0s.
Coverslip slides with DEPEX mounting media and allow to dry in the fume hood before proceeding with microscopy.
Image sections using bright field microscopy for subsequent xenograft characterization.
