Aug 21, 2022

DAB immunostaining of thin, fixed mouse brain tissue sections using HNA or NCAM to characterize human iPSC-derived cell xenografts

DOI
https://dx.doi.org/10.17504/protocols.io.14egn7pb6v5d/v1
  • 1The University of Sydney
Benjamin Trist
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn7pb6v5d/v1
Protocol Citation: Benjamin Trist, Ashish Mathai, Asheeta Prasad 2022. DAB immunostaining of thin, fixed mouse brain tissue sections using HNA or NCAM to characterize human iPSC-derived cell xenografts. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7pb6v5d/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 12, 2022
Last Modified: May 31, 2024
Protocol  Integer ID: 68551
Keywords: NCAM, HNA, Human-to-mouse xenograft, Human iPSC, Immunohistochemistry, ASAPCRN, mouse brain tissue section series, fixed mouse brain tissue section series, mouse brain tissue section, brain of athymic mice, fixed mouse brain tissue section, growth of human ipsc, derived cell, immunohistochemistry, derived cell xenograft, living brain, human ipsc, athymic mice, cell xenografts this protocol, mice
Funders Acknowledgements:
Michael J Fox Foundation
Grant ID: ASAP-000497
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.
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Materials
Equipment:

  • Horizontal rocker
  • Vortex
  • Microcentrifuge
  • Glass petri dish
  • Oven

Consumables:

  • 20 mL scintillation vials
  • Paint brushes
  • Gelatin-Chrom Alum-coating microscope slides
1. See related protocol - Coating superfrost microscope slides with gelatin-chromium potassium sulfate
  • Microscope slide coverslips (no. 1.5, 25 mm x75 mm )
  • Glass pipettes
  • Rubber teats
  • Transfer pipettes

Key reagents:

  • Optimal Cutting Temperature (OCT) compound
  • Bovine Serum Albumin (BSA)
  • Casein
  • Sodium citrate
  • Tween-20 and Triton X-100
  • Ethanol
  • Hydrogen peroxide (H2O2)
  • DEPEX
  • 3, 3'-diaminobenzidine (Sigma #D5905)
  • Antibodies
1 NCAM AbcamCatalog #ab75813
2. HNA (Novus #NOVNBP-313912)
3. Biotinylated anti-rabbit secondary antibody (Vector Labs #BA-1000)
4. Avidin/Biotin HRP ComplexVector LaboratoriesCatalog #PK-6100

Solutions:

  • 1x PBS, pH 7.4
AB
Antigen retrieval (AR) buffer
Sodium citrate2.94 g (10 mM)
Tween-20500 µL (0.05%) 
Up to 1L with dH2O, pH 6.0
  • Quenching solution
10mL (3.3%) 33% H2O2, 50mL (50%) ethanol up to 100mL with 1x PBS
  • 1x PBST
500 µL (0.05%) Tween-20 in 1L 1x PBS
AB
Blocking solution
Casein1 g (1% w/v)
Triton X-100250 µL (0.25% v/v)
Glycine1.5 g (1.5% w/v)
BSA5 g (5% w/v)
Up to 100mL with 1xPBS

Material input (animal, cell, tissue, fraction details):

Thin, fixed athymic mouse brain tissue sections prepared from whole mouse brains grafted with human iPSC-derived neural progenitor cells.



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 00:07:00 (1/3).
7m
Remove anti-freeze solution and wash in 1x PBS at Room temperature for 00:07:00 (2/3).
7m
Remove anti-freeze solution and wash in 1x PBS at Room temperature for 00:07:00 (3/3).
7m
Antigen retrieval (AR)
Incubate sections in AR buffer for 00:30:00 at 70 °C .
30m
Preheat AR buffer to 70 °C prior to addition to sections.
After antigen retrieval, allow sections to cool for 00:30:00 before proceeding with staining.
30m
Perform 3x 7 min washes in 1x PBST with agitation.
Wash in 1x PBST with agitation for 00:07:00 (1/3).
7m
Wash in 1x PBST with agitation for 00:07:00 (2/3).
7m
Wash in 1x PBST with agitation for 00:07:00 (3/3).
7m
Quenching step
Incubate sections in quenching solution for 00:30:00 at Room temperature with gentle agitation.
30m
Perform 3x 7 min washes in 1x PBST with gentle agitation.
Wash in 1x PBST with gentle agitation for 00:07:00 (1/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (2/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (3/3).
7m
Blocking step
Incubate sections in blocking solution for 01:00:00 at Room temperature with gentle agitation.
1h
Primary antibody step
Incubate sections with NCAM (1:20,000) or HNA (1:15,000) primary antibodies diluted in blocking buffer Overnight 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 00:07:00 (1/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (2/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (3/3).
7m
Secondary antibody step
Incubate sections in anti-rabbit biotinylated secondary antibody 1:500 diluted in blocking buffer that has been diluted 2-fold for 02:00:00 at Room temperature .
2h
Perform 3x 7 min washes in 1x PBST with gentle agitation.
Wash in 1x PBST with gentle agitation for 00:07:00 (1/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (2/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (3/3).
7m
Tertiary complex step
Incubate sections in Avidin-Biotin Complex (ABC) kit solution (Vector Laboratories) for 02:00:00 at Room temperature .
2h
Prepare tertiary complex 00:30:00 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).
30m
Perform 3x 7 min washes in 1x PBST with gentle agitation.
Wash in 1x PBST with gentle agitation for 00:07:00 (1/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (2/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (3/3).
7m
Chromogen step
Perform DAB staining as follows;
Prepare DAB solution by dissolving 10 mg DAB tablet into 20 mL PBS (0.5 mg/mL ).
Filter through Whatman paper #1 or 0.22 um syringe filter before use.
Note
NB: DAB is a suspected mutagen and should be handled with care.

Prepare DAB-H2O2 solution immediately prior to use by adding 10 µL 30% H2O2 per 5 mL DAB solution and mix thoroughly.
Incubate sections with DAB-H2O2 solution for 00:08:00 .
8m
Perform 3x 7 min washes in 1x PBST with gentle agitation.

Wash in 1x PBST with gentle agitation for 00:07:00 (1/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (2/3).
7m
Wash in 1x PBST with gentle agitation for 00:07:00 (3/3).
7m
Mount tissue sections onto super-frost slides pre-coated with gelatin-chrome alum and allow to dry at Room temperature Overnight .

Day 3 (2 days later)
33m
Process slide-mounted tissue sections through the following solutions;
dH2O 00:03:00 .
3m
50% ethanol 00:03:00 .
3m
70% ethanol 00:03:00 .
3m
95% ethanol 00:03:00 .
3m
100% ethanol 00:03:00 .
3m
100% ethanol 00:03:00 .
3m
Xylene 00:05:00 .
5m
Xylene 00:05:00 .
5m
Xylene 00:05:00 .
5m
Coverslip slides with DEPEX mounting media and allow to dry in the fume hood Overnight before proceeding with microscopy.

Image sections using bright field microscopy for subsequent xenograft characterization.