Sep 18, 2022

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

DOI
dx.doi.org/10.17504/protocols.io.4r3l275mxg1y/v1
  • 1The University of Sydney
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DOI: 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
Funders Acknowledgement:
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

ReagentAnti-α-synuclein Antibody (211)Santa Cruz BiotechnologyCatalog #sc-12767
ReagentAnti-Ctip2 antibody [25B6] (ab18465)AbcamCatalog #ab18465
ReagentAnti-FOXG1 antibody (ab18259)AbcamCatalog #ab18259
ReagentAnti-NCAM/CD56 Antibody (ERIC 1)Santa Cruz BiotechnologyCatalog #sc-106
ReagentAnti-NeuN AntibodySigma AldrichCatalog #ABN91
ReagentPAX6 Polyclonal AntibodyThermo Fisher ScientificCatalog #42-6600
ReagentHuman/Mouse/Rat SOX2 AntibodyR&D SystemsCatalog #AF2018
ReagentAnti-TBR1 antibodyAbcamCatalog #ab31940
ReagentRabbit 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
ReagentDonkey anti-Mouse IgG (H L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor™ 647Thermo Fisher ScientificCatalog #A-31571
ReagentGoat-anti-rabbit-Alexafluor 488 Thermo Fisher ScientificCatalog #A11008
ReagentAnti-Chicken IgG (H L) highly cross-adsorbed CF594 antibody produced in donkeySigma AldrichCatalog #SAB4600094
ReagentAnti-Goat IgG (H L) highly cross-adsorbed CF™ 488A antibody produced in donkeySigma AldrichCatalog #SAB4600032


Solutions:

  • 1x PBS, Ph7.4

  • Antigen retrieval (AR) buffer
Amount2.94 g (Concentration10 millimolar (mM) ) sodium citrate, Amount500 µL (0.05%) Tween-20, up to Amount1 L with dH2O, Ph6
  • 1x PBST

1. Amount500 µL (0.05%) Tween-20 in Amount1 L 1x PBS

  • Blocking solution

1. Amount1 g (1% w/v) casein, Amount250 µL (0.25% v/v) Triton X-100, Amount1.5 g (1.5% w/v) glycine, Amount5 g (5% w/v) BSA up to Amount100 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)
Day 1 (~3-4 hrs)
Pre-heat oven and Antigen Retrieval (AR) buffer to Temperature70 °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.
Pipetting
Remove anti-freeze solution and perform 3x 7 min washes in 1x PBS at TemperatureRoom 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.


Wash
Remove anti-freeze solution and perform 3x Duration00:07:00 washes in 1x PBS at TemperatureRoom temperature with gentle agitation (1/3).
7m
Remove anti-freeze solution and perform 3x Duration00:07:00 washes in 1x PBS at TemperatureRoom temperature with gentle agitation (2/3).
7m
Remove anti-freeze solution and perform 3x Duration00:07:00 washes in 1x PBS at TemperatureRoom temperature with gentle agitation (3/3).
7m
Antigen retrieval (AR) - Day 1
Antigen retrieval (AR) - Day 1
Incubate sections in pre-heated AR buffer for Duration00:30:00 at Temperature70 °C .

30m
Incubation
After antigen retrieval, allow sections to cool for Duration00:30:00 before proceeding with staining.

30m
Perform 3x 7 min washes in 1x PBST with gentle agitation.

Wash
Perform 3x Duration00:07:00 washes in 1x PBST with gentle agitation (1/3).

7m
Perform 3x Duration00:07:00 washes in 1x PBST with gentle agitation (2/3).
7m
Perform 3x Duration00:07:00 washes in 1x PBST with gentle agitation (3/3).
7m
Blocking step - Day 1
Blocking step - Day 1
Incubate sections in blocking solution for Duration01:00:00 at TemperatureRoom temperature with gentle agitation.

1h
Incubation
Primary antibody step - Day 1
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 DurationOvernight at Temperature4 °C with gentle agitation.

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

Wash
Perform 3 x Duration00:07:00 washes in 1x PBST with gentle agitation (1/3).
7m
Perform 3 x Duration00:07:00 washes in 1x PBST with gentle agitation (2/3).
7m
Perform 3 x Duration00:07:00 washes in 1x PBST with gentle agitation (3/3).
7m
Secondary antibody step
Secondary antibody step
1d 4h 7m
1d 4h 7m
Incubate sections in secondary antibodies, that match the species of chosen antibodies, diluted in blocking buffer for Duration02:00:00 at TemperatureRoom temperature . Incubate in a dark room or cover the vials with foil to protect the fluorophores from light.

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

Wash
Perform 3x Duration00:07:00 washes in 1x PBST with gentle agitation keeping vials protected from the light (1/3).
7m
Perform 3x Duration00:07:00 washes in 1x PBST with gentle agitation keeping vials protected from the light (2/3).
7m
Perform 3x Duration00:07:00 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 TemperatureRoom temperature for Duration01:00:00 - Duration02:00:00 .

3h
Remove ProLong Diamond Antifade mountant from storage at Temperature4 °C and allow the reagent to equilibrate toTemperatureRoom temperature before use.

Coverslip slides with ProLong Diamond Antifade mountant and allow to set at TemperatureRoom temperature for at least Duration24:00:00 in the dark before proceeding with microscopy.

1d
Image sections using fluorescent microscopy for subsequent xenograft characterization.
Imaging
Slides can be stored at Temperature4 °C or Temperature-20 °C .