Oct 11, 2024
Protocol for Free-Floating Immunofluorescence Staining of Mouse Brain Sections
- Cormac Peat1,
- Glenda Halliday1,
- Asheeta Prasad1,
- courtney wright1
- 1University of Sydney
Protocol Citation: Cormac Peat, Glenda Halliday, Asheeta Prasad, courtney wright 2024. Protocol for Free-Floating Immunofluorescence Staining of Mouse Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rpdjg1b/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 12, 2024
Last Modified: October 11, 2024
Protocol Integer ID: 107453
Keywords: Immunofluorescence, mouse brain, alpha-synuclein, astrocytes, transgenic, A53T, free floating, ASAP, MJFF
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol outlines the free floating immunofluorescence staining protocol for the detection of astrocytes, functional astrocyte markers and alpha-synuclein deposits in brain samples derived from 9 month old transgenic A53T mice.
Guidelines
Carry out all procedures in minimal light conditions to minimise signal bleaching.
Materials
Materials
Antibodies:
- GFAP: GFAP Polyclonal Antibody,Thermo #PA110004, Chk, IgY, 1:1000
- S100B: S100B Polyclonal Antibody, R&D Systems #AF1820, Gt, IgG, 1:1000
- ALDH1L1: ALDH1L1 Polyclonal Antibody, Thermo #PA578622, Rbt, IgG, 1:1000
- Alpha Synuclein: Alpha-synuclein Monoclonal Antibody, BD Transduction Laboratories #610787, IgG1, 1:1000
- CYP27B1: CYP27B1 Polyclonal Antibody, Sigma #ABN182, Rbt, , 1:1000
- VEGFA: VEGFA Polyclonal Antibody, GeneTex #GTX102643, Rbt, IgG, 1:1000
| A | B | C | |
| Marker | Primary antibodies | Secondary antibodies | |
| GFAP | chicken anti-GFAP (Thermo #PA110004 1:1000) | Goat anti-chicken 488 (Thermo #A11039; 1:250) or Goat anti-chicken 594 (Thermo #A11042; 1:250) or Goat anti-chicken 647 (Thermo #A21449; 1:250) | |
| S100b | goat anti-S100b (R&D Systems #AF1820; 1:1000) | Donkey anti-goat 647 (Thermo #A21447; 1:250) or Donkey anti-goat 594 (Thermo #A11058; 1:250) | |
| ALDH1L1 | rabbit anti-ALDH1L1 (Thermo #PA578622; 1:1000) | Goat anti-rabbit 647 (Thermo #A32733; 1:250) or Goat anti-rabbit 594 (Thermo #A11012; 1:250) | |
| a-synuclein | Mouse anti-alpha-synuclein (BD #610787; 1:1000) | Goat anti-mouse 488 (Thermo #A11001; 1:250) | |
| VEGFA | Rabbit anti-VEGFA (Genetex #GTX102643;1:1000) | Goat anti-rabbit 594 (Thermo #A11012; 1:250) | |
| CYP27B1 | Rabbit anti-CYP27B1 (Merck #ABN182; 1:1000) | Goat anti-rabbit 594 (Thermo #A11012; 1:250) |
Equipment:
- Orbital Shaker
- Petri Dish
- Paintbrushes
Consumables:
- Superfrost + microscope slides
- Microscope slide coverslips (no. 1.5 thickness, 22x50mm)
- 6-well cell culture plates with mesh inserts
- Scintillation vials
- 30G x 1/2" 0.3mm x 13mm Hypodermic Needles
Key Reagents:
- Blocking Buffer: 4% BSA (w/v), 1% casein (w/v), 1.5% glycine (w/v), 0.25% Triton X-100 (v/v) in 1X PBS
- Triton X-100 (For 1X TBST 0.25%)
- Hoechst counterstain dye (Thermo #62249, 1ug/mL)
- Prolong Diamond Mounting Medium (Invitrogen #P36970)
Safety warnings
For hazard information and safety warnings, refer to the relevant SDS (Safety Data Sheet).
Ethics statement
Procedures involving the use of animals must receive approval from relevant animal ethics committees and must comply with institutional and national regulations
Before start
Sections described in this protocol are 30um free floating mouse brain sections, fixed with 4% PFA and cryopreserved in 30% sucrose in PBS. Prior to this procedure, sections are stored in antifreeze at -20 °C
Outline
Outline
Mouse brain tissue sections are washed initially to remove cryoprotectant, then blocking and primary antibody incubation overnight are carried out. Further washing is then followed by secondary antibody incubation and counterstaining, then the sections are mounted to microscope slides and coverslipped.
Tissue Preparation
Tissue Preparation
20m
20m
1. Sections stored in a -20 °C freezer are removed and allowed to equilibrate to room temperature for 00:15:00 . .
2. Transfer sections into a mesh insert within a 6-well cell culture plate to separate them from the storage solution.
3. Move the mesh insert to the adjacent well containing approx. 5 mL 1X TBST 0.25% . Wash 3x with this solution for 00:05:00 per wash at Room temperature , changing solutions each wash. Agitate gently using an orbital shaker.
20m
Blocking
Blocking
1h
1h
1. Transfer sections from the final wash well into a new well containing IF blocking buffer and incubate at Room temperature for 01:00:00 on the orbital shaker on low speed
1h
Primary Antibody Incubation
Primary Antibody Incubation
1. Prepare appropriate antibody solutions in scintillation vials by diluting primary antibody in IF blocking buffer. Prepare approx. 2 mL per vial .
2. Transfer sections from cell culture plate to the vial with appropriate antibody solution.
3. Place vial on orbital shaker and agitate lightly Overnight at Room temperature . Cover vial such that it is sheilded from light.
Secondary Antibody Incubation
Secondary Antibody Incubation
2h 10m
2h 10m
**This and subsequent steps should be carried out in a darkened room if possible to avoid exposure to light.
1. Transfer sections from vial into well insert within a 6 well culture plate, and separate them from antibody solution.
2. Move the mesh insert to the adjacent well containing approx. 5 mL 1X TBST 0.25% . Wash 3x with this solution for 00:05:00 per wash at Room temperature , changing solutions each wash. Agitate gently using an orbital shaker.
3. Prepare new scintillation vial with appropriate secondary antibody solution, diluted in IF blocking buffer. Prepare approx. 2 mL per vial . Ensure the vial and solution are obscured from light.
4. Transfer sections from well insert to appropriate vial and incubate at Room temperature for 03:00:00 Agitate gently with orbital shaker and ensure vial is shielded from light.
5. Following incubation, transfer sections from vial into well insert and carry out 3x 00:05:00 washes with TBST 0.25% at Room temperature
3h 10m
Counterstain
Counterstain
1. Prepare approx. 2 mL of 1ug/mL solution of Hoechst diluted in IF blocking buffer in a scintillation vial.
2. Transfer sections from well insert to vial and incubate for exactly 00:05:00
3. Place sections back into well insert and carry out 3x 00:05:00 washes with TBST 0.25% at Room temperature
10m
Mounting and Coverslipping
Mounting and Coverslipping
1d 0h 10m
1d 0h 10m
1. Transfer sections from well insert into a petri dish containing 1X TBST 0.25%.
2. Take a microscope slide and insert it into the dish at an approx. 45° angle, allowing most of the surface of the slide to be submerged.
3. Using a fine paintbrush, gently move one section at a time onto the slide surface and orient it correctly. Ensure the tissue is flat with no folds. Hold each section in place and tilt the slide gently to remove excess solution and minimise slippage.
4. Repeat this process until all the sections are mounted. Allow all to dry for approx. 00:10:00
5. After slides have dried, apply 2-3 drops of Prolong Diamond Mounting Medium. Place a coverslip at one end and gently lower the other end of the coverslip with a 30G x 1/2" 0.3mm x 13mm Hypodermic Needle (or similar), ensuring no air bubbles form underneath. Gently press down to remove excess mounting medium.
6. Place slides in a slide box with the top of slide/coverslip/sections facing up and allow to dry for minimum 24:00:00 , or until mounting medium has completely hardened.
1d 0h 10m
Protocol references
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