Nov 16, 2025

Immunofluorescence in PFA-fixed grafted mouse brain slices 

DOI
https://dx.doi.org/10.17504/protocols.io.3byl46woogo5/v1
  • 1Karolinska Institute Stockholm
  • SOX6 mDA differentiation
Roberto Garcia Swinburn
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl46woogo5/v1
Protocol Citation: Roberto Garcia Swinburn, Ernest Arenas 2025. Immunofluorescence in PFA-fixed grafted mouse brain slices . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46woogo5/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 03, 2025
Last Modified: November 16, 2025
Protocol  Integer ID: 226317
Keywords: immunohistochemistry, immunofluorescence, animal tissue, immunofluorescence in pfa, grafted mouse brain slice, fixed grafted mouse brain slice, µm mouse brain slice, conjugated antibody, immunofluorescence, double labeling of protein, fixed brain slice, protein expression, protein, brain slice, pfa, succesful on pfa, using fluorophore
Abstract
This protocol was used to tag protein expression using fluorophore-conjugated antibodies on 20 µm mouse brain slices. Double labeling of proteins and nuclei was succesful on PFA-fixed brain slices.
Materials
PBS1X
PBTx0.3% [Triton-X 0.3% in PBS]
PBTx0.1% [Triton-X 0.1% in PBS]
Blocking Serum Solution (BSS) [Donkey Serum 5%, BSA 1 mg/ml, PBTx0.1%]
Primary antibodies
Secondary antibodies
ReadyProbes Autofluorescence quenching agent
DAPI
Mounting medium

Protocol materials
FluoromountMerck MilliporeSigma (Sigma-Aldrich)Catalog #F4680
Day one: Primary antibody
19h 35m
Place up to eight 20-30 µm slices in wells filled with PBS1X.
For free-floating, 12 well plates were used and slow constant shaking to ensure coating without compromising sample structural integrity.

Wash tissue with PBS1X.
PBS1X can be commercial or self-made, but make sure that it is 7.4

5m
Wash tissue with PBS1X
5m
Wash with PBTx0.3%
15m
Wash with PBTx0.1%
5m
Wash with PBTx0.1%
5m
Incubate samples in BSS [Donkey Serum 5%, BSA 1 mg/ml, PBTx0.1%], preferably in plates of smaller wells (e.g 24 well) to optimize volume used.
Blocking solution was prepared for secondary (2ndary) antibodies hosted in donkey. If 2ndary antibody is hosted in goat, add Goat serum 5%. Remember to not use donkey or goat serum if the primary antibody is hosted on any of those serums.
1h
Incubate samples in primary antibody solution (in BSS) at 4 °C
Always remember to check antibody host. Use recommended dilution, if no recommendations are there for immunohistochemistry, rule of thumb is 5 x recommendation of immunocytochemistry.
18h
Day two: Secondary antibody
2h 50m
Recover slices to 12 well plates. Wash with PBTx0.1%
5m
Wash with PBTx0.1%
5m
Incubate in secondary solution (in BSS) Room temperature

2h
Wash tissue with PBS1X
5m
Wash tissue with PBS1X
5m
Optional: Incubate samples in ReadyProbes Tissue Autofluorescence quencher (Thermo Scientific, R37630).
10m
Wash with PBS1X
5m
Incubate in DAPI:PBS 1:1000 solution
10m
Wash with PBS1X
5m
Mount on glass slides with Fluorescent Mounting Media FluoromountMerck MilliporeSigma (Sigma-Aldrich)Catalog #F4680