Apr 24, 2025
Immunofluorescence protocol for floating mouse brain sections
- Mary Alice Allnutt1
- 1Yale University
Protocol Citation: Mary Alice Allnutt 2025. Immunofluorescence protocol for floating mouse brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx31ddg8j/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: November 01, 2023
Last Modified: April 24, 2025
Protocol Integer ID: 90932
Keywords: ASAPCRN, floating mouse brain section, mouse brain sections this protocol, floating section, immunofluorescence protocol, immunofluorescence
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000529
Abstract
This protocol details the immunofluorescence for floating sections.
Attachments
Materials
Some of our preferred antibodies:
| A | B | C | D | |
| Host | Target | Catolog No. | Recommended Dilution (Brain tissue) | |
| Rabbit | pSer129 alpha-synuclein | Abcam ab51253 | 0.59722 | |
| Mouse | Alpha-synuclein | BD Biosciences 610786 | 0.38889 | |
| Guinea pig | GFAP | Synaptic systems 173 004 | 0.31944 | |
| Rabbit | Iba1 | Wako (019-19741) | 0.25 | |
| Goat* | ChAT | Millipore AB144P | 0.38889 |
*use donkey serum and donkey-hosted secondary antibodies for all antibodies in an experiment with this primary
Recombinant Anti-Alpha-synuclein (phospho S129) antibodyAbcamCatalog #ab51253
BD Transduction Laboratories™ Purified Mouse Anti-α-SynucleinBD BiosciencesCatalog #610786
GFAP antibodySynaptic SystemsCatalog #173 004
IBA AbWakoCatalog #019-19741
Goat anti-ChAT antibodyMerck Millipore (EMD Millipore)Catalog #AB144P
Troubleshooting
Sectioning and long-term tissue storage
30m
Section the brains at 30 µm thickness on Leica cryostat (CM1860).
Collect the sections in cryoprotectant solution in 12-well plates and store at -20 °C .
| A | B | C | |
| a | Sucrose | 300 g | |
| b | Polyvinyl-pyrrolidone (PVP-40) | 10 g | |
| c | 0.1M PB | 500 ml | |
| d | Ethylene glycol | 300 ml |
e. Add PVP-40 to 0.1M PB (very slowly- this will take a while to dissolve). Stir to dissolve. Slowly add the sucrose to dissolve, and then add the ethylene glycol and bring the final volume to 1000 ml with 0.1M PB. Store at -20°C.
Wash the sections that are stored in cryoprotectant 3x 10 min prior to beginning and immunostaining protocols.
Wash the sections that are stored in cryoprotectant for 00:10:00 prior to beginning and immunostaining protocols (1/3).
10m
Wash the sections that are stored in cryoprotectant for 00:10:00 prior to beginning and immunostaining protocols (2/3).
10m
Wash the sections that are stored in cryoprotectant for 00:10:00 prior to beginning and immunostaining protocols (3/3).
10m
Immunofluorescence protocol for free-floating sections
8h 6m
Note
Wash buffer: TBS + 0.1% Triton x-100 (Tx)
Equilibration: TBS + 0.1% Tx, 00:10:00 .
10m
Penetration: TBS + 0.5% Tx, 00:20:00 .
20m
Wash 3x 2 min.
Wash for 00:02:00 (1/3).
2m
Wash for 00:02:00 (2/3).
2m
Wash for 00:02:00 (3/3).
2m
Neutralization: 0.3 Molarity (M) Glycine in TBS + 0.1% Tx, 00:30:00 .
30m
Wash 3x 2 min.
Wash for 00:02:00 (1/3).
2m
Wash for 00:02:00 (2/3).
2m
Wash for 00:02:00 (3/3).
2m
Blocking: 3% goat serum in TBS + 0.1% Tx, 01:20:00 .
1h 20m
Wash 3x 2 min.
Wash for 00:02:00 (1/3).
2m
Wash for 00:02:00 (2/3).
2m
Wash for 00:02:00 (3/3).
2m
Primary incubation:
Prepare in 1% goat serum in TBS + 0.1% Tx.
Overnight 4°C, shaking.
1h 20m
Wash 3-4x 2 min.
Wash for 00:02:00 (1/4).
2m
Wash for 00:02:00 (2/4).
2m
Wash for 00:02:00 (3/4).
2m
Wash for 00:02:00 (4/4).
2m
Secondary incubation:
Prepare in 1% goat serum in TBS + 0.1% Tx.
1:500 for 04:00:00 at Room temperature .
4h
Wash 3-4x 2 min with TBS + 0.1% Tx and then twice with TBS alone.
Wash for 00:02:00 with TBS + 0.1% Tx and then twice with TBS alone (1/4).
2m
Wash for 00:02:00 with TBS + 0.1% Tx and then twice with TBS alone (2/4).
2m
Wash for 00:02:00 with TBS + 0.1% Tx and then twice with TBS alone (3/4).
2m
Wash for 00:02:00 with TBS + 0.1% Tx and then twice with TBS alone (4/4).
2m
Quench autofluorescence (optional):
Dip sections briefly in ultrapure H2O.
Incubate in CuSO4 buffer (10 millimolar (mM) CuSO4 in 50 millimolar (mM) ammonium acetate buffer, 5 ) for 00:15:00 at Room temperature .
15m
Dip sections in ultrapure water again briefly prior to mounting.
Note
Sections may appear slightly wrinkled after this treatment, but should still be able to be mounted without issue.
Mounting
Fill petri dish with TBS. Have serological pipette and secondary container available.
Place a clean slide in petri dish diagonally, with label resting against the side of the dish, above the level of the solution, and the rest submerged.
Using fine detail paint brush, lift floating section and gently adhere to slide near the top of the TBS. Can gently tap the section to get it to stick to the slide, or lift one corner of the section along the slide slightly above the water level to help it stick.
Using serological pipette, gently remove a few mL of TBS, leaving the section adhered to the slide.
After all sections are mounted, allow excess TBS to dry and then mount using Vectashield mounting medium with DAPI and a glass coverslip.
Note
Can replace TBS with PBS if not using antibodies targeting phosphorylated proteins.