Aug 17, 2023

Public workspaceFree floating immunofluorescent staining protocol on mouse brain sections V.2

DOI
dx.doi.org/10.17504/protocols.io.j8nlkoo55v5r/v2
  • 1University of Sydney
courtney.wright Wright
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkoo55v5r/v2
Protocol Citation: Giselle Sagredo, YuHong Fu, Hongyun Li 2023. Free floating immunofluorescent staining protocol on mouse brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkoo55v5r/v2Version created by courtney.wright Wright
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 17, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86591
Keywords: ASAPCRN, immunofluorescence, tyrosine hydroxylase, phospho-Serine129, alpha synuclein, mouse brain, iPSC derived cells, free floating
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol describes our free-floating immunofluorescence staining protocol used to investigate the fate and pathology of human iPSC-derived cells grafted in the mouse brain. This protocol allows for the post-mortem visualisation and analysis of the morphology and pathological inclusions of the transplanted human iPSC-derived cells carrying different PD-related mutations in different regions within mouse brain tissue sections.
Guidelines
IMPORTANT: perform all antibody incubation steps and steps following in minimal light so as not to bleach signals prior to imaging
Materials
Antibodies
  • TH(IgG2b): TH Monoclonal Antibody (OTI3G3),TrueMAB™ #TA506549, Ms, IgG2b, clone OTI3G3, 1:200
https://www.thermofisher.com/antibody/product/TH-Antibody-clone-OTI3G3-Monoclonal/TA506549
  • Syn204(IgG2a): Anti-α-Synuclein Antibody, Biolegend #838201, Ms, IgG2a, clone Syn204 (aa 87-110) 1:1, 1:200
https://www.biolegend.com/en-us/products/anti-alpha-synuclein-antibody-10995?GroupID=BLG15651
ABCDE
Comb#1 Primary TH IgG2b Syn204 S129
Cat #  TA506549, MS IgG2b 838201, Ms IgG2a, 1:1 ab51253, Rb
dilution    200  200 500 
Comb#1 Secondary  Goat @ mouse IgG2b 647  Goat @ mouse IgG2a 568  Donkey @ rabbit 488 Hoechst33342
Cat #    A-21242   A-21134 A-21206
dilution   1:250 1:250 1:200 1:1000
Equipment
  • Orbital shaker
  • black porcelain spot plate

Consumables
  • microscope slides
  • 6-well plates and net inserts
  • Microscope slide coverslips (no. 1.5 thickness, 22x50mm)

Key reagents
  • Blocking buffer for IF in tissue section:  2% Donkey serum, 1%BSA, 0.2% TritonX-100, 0.1% gelatine, 0.1% Tween-20 in 1XPBS
  • Citrate buffer (0.01M pH 6.0): 2.94g/L tri-sodium citrate (dihydrate) in deionised water
  • sodium borohydride
  • Tween-20 and Triton X-100
  • DAKO Fluorescence Mounting Medium

Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
NOTE: Sodium borohydride is highly toxic and flammable
Experimental outline
Experimental outline
Briefly, the mouse brain tissue sections are prepared by washing off the cryoprotectant medium and then antigen retrieval is performed followed by quenching, blocking and primary antibody incubation. Sections are then washed and incubated in the appropriate secondary antibody solution and are then mounted, cover-slipped and sealed.
Day 1 - Tissue preparation
Day 1 - Tissue preparation
30 um mouse brain sections were stored in anti-freeze solution at Temperature-20 °C until required.
  1. Remove samples from freezer and equilibrate at TemperatureRoom temperature for Duration00:10:00 - Duration00:20:00
  2. Pour sections into a well insert in a 6-well plate to separate storage solution from section
  3. Move the well insert to another well containing approximately Amount6 mL of 1x PBS. Wash at least 5x with 1x PBS for Duration00:05:00 each on an orbital shaker using low speed at TemperatureRoom temperature
35m
Antigen retrieval
Antigen retrieval
  1. Incubate the sections in 10mM sodium citrate buffer (pH 6.0) for Duration00:30:00 . Let it cool to TemperatureRoom temperature
  2. Rinse the sections 3xDuration00:05:00 each in 1X PBS
35m
Quenching aldehyde group
Quenching aldehyde group
  1. Weigh NaBH4 to make 0.1~0.5% in 1X PBS, made fresh
  2. Move the insert with sections into the fresh-made solution for Duration00:30:00 at TemperatureRoom temperature
  3. Wash 2x Duration00:05:00 in 1X PBS
35m
Blocking
Blocking
  1. Incubate sections in normal donkey serum IF blocking bufferDuration02:00:00 TemperatureRoom temperature on shaker 60 rpm
2h
Primary antibody incubation
Primary antibody incubation
Make primary antibody cocktails in blocking buffer
  1. Prepare ~Amount300 µL per sample of primary antibody solution consisting of selected primary antibody (diluted appropriately) in home-made normal donkey serum IF blocking buffer
  2. Transfer sections from well insert into wells of black porcelain spot plate containing primary antibody solution to bind to the antigen(s) of interest
  3. Place the plate on a rotating mixer using low speed (speed 7 rpm) and incubate Duration72:00:00 at Temperature4 °C (or 3X night/ over weekend)
3d
Day 2 - Secondary antibodies
Day 2 - Secondary antibodies
  1. The following day, pour sections into a well insert in a 6-well plate to separate sections from primary antibody solution.
  2. Wash sections 3 times with 1x PBST at TemperatureRoom temperature . Note: Duration00:00:30 for the first two rinses, 3x Duration00:10:00 for additional washing
  3. Prepare Amount300 µL per sample of secondary antibody solution consisting of appropriate secondary antibody + Hoechst 33342 (diluted accordingly) in blocking buffer (shield solution from light)
  4. Transfer sections into the black porcelain spot plate containing Amount300 µL secondary antibody cocktail
  5. Incubate for Duration02:00:00 at TemperatureRoom temperature on orbital shaker using low speed (shield solution from light).
  6. Pour sections into a well-insert in a 6-well plate containing 1X PBST to separate sections from the secondary antibody solution
  7. Continuing to shield samples from light, wash 3 times with 1x PBS for Duration00:05:00 at TemperatureRoom temperature
2h 15m 30s
Mounting
Mounting
  1. Pour sections into a glass petri dish
  2. Submerge a glass slide into the 1x PBS and use a fine paintbrush to coax the sections towards the slide
  3. Gently tap the sections onto the slide, making sure there are no wrinkles or folds
  4. Repeat until all sections are mounted onto the slide(s)
Cover-slipping
Cover-slipping
15m
15m
  1. After sections are dried onto the slide(s), about Duration00:15:00 at TemperatureRoom temperature or until sections look opaque (remember to shield slides from light), apply an appropriate aqueous mounting medium (hardening or non-hardening). Antifading (DAKO Fluorescence Mounting Medium is preferred if using a fluorescent conjugated secondary antibody
  2. Using tweezers, place a coverslip on top of the medium. Cover with filter paper and press down firmly to remove excess mounting medium
  3. Image sections using an appropriate microscope. Store in a dark slide box at Temperature4 °C



15m