Feb 18, 2024

Public workspaceFree floating immunofluorescence protocol on mouse brain sections for tau pathology

DOI
dx.doi.org/10.17504/protocols.io.6qpvr3j23vmk/v1
  • 1University of Sydney
courtney.wright Wright
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr3j23vmk/v1
Protocol Citation: Felicia Suteja, Hongyun Li, YuHong Fu 2024. Free floating immunofluorescence protocol on mouse brain sections for tau pathology. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3j23vmk/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: February 18, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95382
Keywords: ASAPCRN
Funders Acknowledgements:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol describes our free-floating multiplexed immunofluorescent staining protocol to ascertain levels of tau and phospho- tau in mouse tissue from transplanted human iPSC cells carrying different PD related mutations.
Guidelines
IMPORTANT: perform all antibody incubation steps and steps following in minimal light so as not to bleach signals prior to imaging
Materials
Equipment
  • Orbital shaker
  • black porcelain spot plate
Consumables
  • microscope slides
  • 6-well plates and net inserts
  • Microscope slide coverslips (no. 1.5 thickness, 22x50mm)
1st AT8(Ms IgG1) SP70 (rb) TH (Ms IgG2b)
Cat# Thermofisher #MN1020 Sigma #SAB5500182 Thermofisher # TA506549
dilution 200 (400 if 1:1 with glycerol) 200 200
Primary antibody details
ABCDE
2nd Gt @ Ms IgG1 Dn@Rb-AF568 Gt@Ms IgG2b-AF647 Hoechst 33,342 (1mg/ml stock)
Cat# ThermoFisher #A-21206 ThermoFisher #A-10042 Thermofisher #A-#A-21242 Sigma #B2261
dilution 250 250 200 1:1000
Secondary antibody details


Homemade IF blocking buffer (NDS)
• 2% Donkey serum (Sigma, D9663)
• 1% BSA (best with IgG-free and protease-free) (Sigma, A9085 or JIR #001-000-173). 
• 0.2% TritonX-100 (Sigma, T9284).  
• 0.1% gelatine (from fish skin, Sigma, G7041).  
• 0.1% Tween-20 (Sigma, P1379).  
• 0.01% Sodium Azide (Sigma, S2002)
in 1XPBS, aliquoted and store at -20°C.
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).

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 prep
Day 1 - Tissue prep
5m
5m
30 μm mouse brain sections were stored in anti-freeze solution at Temperature-20 °C until required.

  1. Pour sections into a well insert in a 6-well plate to separate storage solution from section
  2. Move the well insert to another well containing approximately Amount6 mL of 1x PBS. Wash at least 6x with 1x PBS for Duration00:05:00 each on an orbital shaker using low speed at

5m
Antigen retrieval
Antigen retrieval
27m
27m
  1. Place sections in labelled glass vials containing Amount6 mL 1x citric buffer (CB) pH6.0.
  2. Place in the steamer (Breville, Model: BFS800BSS) on high for Duration00:22:00 Temperature98 °C .
  3. Cool down to TemperatureRoom temperature
  4. Place the sections back into its corresponding wells.
  5. 1xPBS wash: 2 x Duration00:05:00
27m
Blocking and primary incubation
Blocking and primary incubation
3d 2h
3d 2h
  1. Incubate in blocking buffer: Duration02:00:00 TemperatureRoom temperature on shakerShaker60 rpm
2. Make primary antibody cocktail in home-made IF buffer
3. Label ceramic plate and place sections in antibody cocktail: Duration72:00:00 at Temperature4 °C on the shaker
- AT8(Ms IgG1), Thermofisher #MN1020 1:200
- SP70 (rb), Sigma #SAB5500182 1:200
- TH (Ms IgG2b), Thermofisher # TA506549 1:200
3d 2h
Day 4 - Secondary antibodies
Day 4 - Secondary antibodies
2h 25m
2h 25m
  1. Transfer sections from ceramic plate to well plates
  2. 0.1% PBST wash: 3 xDuration00:10:00
  3. Make secondary cocktails as below in IF blocking buffer
- Gt @ Ms IgG, ThermoFisher #A-21206 1:250
- Dn@Rb-AF568, ThermoFisher #A-10042 1:250
- Gt@Ms IgG2b-AF647, Thermofisher #A-#A-21242 1:200
- Hoechst 33,342 (1mg/ml stock) Sigma #B2261 1:1000
4. Place sections and antibody cocktails in a ceramic plate: Duration02:00:00 TemperatureRoom temperature on the shaker Shaker60 rpm
5. Place sections back into wells
6. 0.1% PBST wash: 3 x Duration00:10:00
7. 1x PBS wash: 3 x Duration00:05:00
2h 25m
Mounting and sealing
Mounting and sealing
5m
5m
  1. Label slides with corresponding section and antibody information
  2. Place sections in a petri dish with 1xPBS and nudge the sections onto slides with brush
  3. Mount with anti-fade media (DAKO Fluorescence Mounting Medium, Agilent, cat# S302380-2)
  4. Wait for sections to become dry and seal with sealant (Biotium CoverGripTM Coverslip Sealant, cat#23005)
  5. Store in slide box and store in fridge/cool room Duration00:05:00

5m