Sep 19, 2022

Multiplex fluorescent immunostaining of thin, fixed mouse brain tissue sections to characterize human iPSC-derived cell xenografts

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l275mxg1y/v1
  • 1The University of Sydney
Benjamin Trist
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l275mxg1y/v1
Protocol Citation: Benjamin Trist, Louise Cottle 2022. Multiplex fluorescent immunostaining of thin, fixed mouse brain tissue sections to characterize human iPSC-derived cell xenografts. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l275mxg1y/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: September 08, 2022
Last Modified: May 31, 2024
Protocol  Integer ID: 69719
Keywords: Human iPSC, Immunohistochemistry, Fluorescent, Human-to-mouse xenograft, ASAPCRN, fixed mouse brain tissue section series, mouse brain tissue section series, multiplex fluorescent immunohistochemistry protocol, fixed mouse brain tissue section, mouse brain tissue section, maturation of human ipsc, brain of athymic mice, brain tissue, human ipsc, derived cell, derived cell xenograft, immunohistochemistry, cell xenografts this protocol, living brain, ipsc, cell, mice, athymic mice
Funders Acknowledgements:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol describes our multiplex fluorescent immunohistochemistry protocol used 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, growth and maturation 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:

  • 20mL 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 thickness, 22x50mm)
  • 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
  • ProLong Diamond Antifade mountant


Primary antibodies:

ABCD
TargetSpeciesDilutionCompany, Catalog #
alpha-SynucleinMouse1:1000Santa Cruz, sc-12767
CTIPRat1:1000Abcam, ab18465
FOXG1Rabbit1:500Abcam, ab18259
HNAMouse1:1000Novus, #NOVNBP-313912
NCAM/CD56 (ERIC-1)Mouse1:100Santa Cruz, sc-106
NeuNChicken1:1000Merck-Millipore, ABN91
Pax6Rabbit1:1000ThermoFisher Scientific, #42-660
SOX2Mouse1:500R&D Systems, AF2018
Tbr1Rabbit1:1000Abcam, ab31940
Tyrosine HydroxylaseRabbit1:1500Pel-freeze, P40101-0

Anti-α-synuclein Antibody (211)Santa Cruz BiotechnologyCatalog #sc-12767
Anti-Ctip2 antibody [25B6] (ab18465)AbcamCatalog #ab18465
Anti-FOXG1 antibody (ab18259)AbcamCatalog #ab18259
Anti-NCAM/CD56 Antibody (ERIC 1)Santa Cruz BiotechnologyCatalog #sc-106
Anti-NeuN AntibodyMerck MilliporeSigma (Sigma-Aldrich)Catalog #ABN91
PAX6 Polyclonal AntibodyThermo Fisher ScientificCatalog #42-6600
Human/Mouse/Rat SOX2 AntibodyR&D SystemsCatalog #AF2018
Anti-TBR1 antibodyAbcamCatalog #ab31940
Rabbit Tyrosine HydroxylasePel-FreezCatalog #P40101-0


Secondary antibodies:

ABCD
TargetSpeciesDilutionCompany, Catalog #
anti-mouse AF647Donkey1:500Life Technologies, A31571
anti-rabbit AF647Goat1:500Life Technologies, A11008
anti-chicken CF594Goat1:500Merck, SAB4600094
anti-goat CF488ADonkey1:500Merck, SAB4600032
Donkey anti-Mouse IgG (H L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor™ 647Thermo Fisher ScientificCatalog #A-31571
Goat-anti-rabbit-Alexafluor 488 Thermo Fisher ScientificCatalog #A11008
Anti-Chicken IgG (H L) highly cross-adsorbed CF594 antibody produced in donkeyMerck MilliporeSigma (Sigma-Aldrich)Catalog #SAB4600094
Anti-Goat IgG (H L) highly cross-adsorbed CF™ 488A antibody produced in donkeyMerck MilliporeSigma (Sigma-Aldrich)Catalog #SAB4600032


Solutions:

  • 1x PBS, 7.4

  • Antigen retrieval (AR) buffer
2.94 g (10 millimolar (mM) ) sodium citrate, 500 µL (0.05%) Tween-20, up to 1 L with dH2O, 6
  • 1x PBST

1. 500 µL (0.05%) Tween-20 in 1 L 1x PBS

  • Blocking solution

1. 1 g (1% w/v) casein, 250 µL (0.25% v/v) Triton X-100, 1.5 g (1.5% w/v) glycine, 5 g (5% w/v) BSA up to 100 mL with 1x PBS

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 (~3-4 hrs)
Pre-heat oven and Antigen Retrieval (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.
Note
  • 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 perform 3x 00:07:00 washes in 1x PBS at Room temperature with gentle agitation (1/3).
7m
Remove anti-freeze solution and perform 3x 00:07:00 washes in 1x PBS at Room temperature with gentle agitation (2/3).
7m
Remove anti-freeze solution and perform 3x 00:07:00 washes in 1x PBS at Room temperature with gentle agitation (3/3).
7m
Antigen retrieval (AR) - Day 1
Incubate sections in pre-heated AR buffer for 00:30:00 at 70 °C .

30m
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 gentle agitation.

Perform 3x 00:07:00 washes in 1x PBST with gentle agitation (1/3).

7m
Perform 3x 00:07:00 washes in 1x PBST with gentle agitation (2/3).
7m
Perform 3x 00:07:00 washes in 1x PBST with gentle agitation (3/3).
7m
Blocking step - Day 1
Incubate sections in blocking solution for 01:00:00 at Room temperature with gentle agitation.

1h
Primary antibody step - Day 1
Incubate sections with desired primary antibody combinations (to characterise either cortical or ventral midbrain derived xenografts) diluted in blocking buffer Overnight at 4 °C with gentle agitation.

1h
Day 2 (~4hrs)
Perform 3 x 7 min washes in 1x PBST with gentle agitation.

Perform 3 x 00:07:00 washes in 1x PBST with gentle agitation (1/3).
7m
Perform 3 x 00:07:00 washes in 1x PBST with gentle agitation (2/3).
7m
Perform 3 x 00:07:00 washes in 1x PBST with gentle agitation (3/3).
7m
Secondary antibody step
1d 4h 7m
Incubate sections in secondary antibodies, that match the species of chosen antibodies, diluted in blocking buffer for 02:00:00 at Room temperature . Incubate in a dark room or cover the vials with foil to protect the fluorophores from light.

2h
Perform 3x 7 min washes in 1x PBST with gentle agitation keeping vials protected from the light.

Perform 3x 00:07:00 washes in 1x PBST with gentle agitation keeping vials protected from the light (1/3).
7m
Perform 3x 00:07:00 washes in 1x PBST with gentle agitation keeping vials protected from the light (2/3).
7m
Perform 3x 00:07:00 washes in 1x PBST with gentle agitation keeping vials protected from the light (3/3).
7m
Mount tissue sections in a dimly lit room (to protect the fluorophores) onto super-frost slides pre-coated with gelatin-chrome alum and allow to dry in the dark at Room temperature for 01:00:00 - 02:00:00 .

3h
Remove ProLong Diamond Antifade mountant from storage at 4 °C and allow the reagent to equilibrate toRoom temperature before use.

Coverslip slides with ProLong Diamond Antifade mountant and allow to set at Room temperature for at least 24:00:00 in the dark before proceeding with microscopy.

1d
Image sections using fluorescent microscopy for subsequent xenograft characterization.
Slides can be stored at 4 °C or -20 °C .