Nov 17, 2025

DAB staining in PFA-fixed grafted mouse brain slices 

 Forked from Immunofluorescence in PFA-fixed grafted mouse brain slices
DOI
https://dx.doi.org/10.17504/protocols.io.rm7vz9585gx1/v1
  • 1Karolinska Institute Stockholm
  • SOX6 mDA differentiation
Roberto Garcia Swinburn
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vz9585gx1/v1
Protocol Citation: Roberto Garcia Swinburn, Ernest Arenas 2025. DAB staining in PFA-fixed grafted mouse brain slices . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9585gx1/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 11, 2025
Last Modified: November 17, 2025
Protocol  Integer ID: 227002
Keywords: immunohistochemistry, immunofluorescence, animal tissue, dab labeling of protein, grafted mouse brain slice, fixed grafted mouse brain slice, dab labeling, µm mouse brain slice, conjugated antibody, protein expression, fixed brain slice, protein, dab, brain slice
Abstract
This protocol was used to tag protein expression using HRP-conjugated antibodies on 20 µm mouse brain slices. DAB 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
3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905
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 [Goat serum 5%, BSA 1 mg/ml, PBTx0.1%], preferably in plates of smaller wells (e.g 24 well) to optimize volume used.
If 2ndary antibody is hosted in donkey, use donkey serum
1h
Incubate samples in primary antibody solution (in BSS) at 4 °C
We used mouse-anti STEM121 for our protocol.
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 HRP-conjugated secondary solution (in BSS) Room temperature
We used Goat anti-mouse
2h
Wash with PBTx0.1%
5m
Wash with PBTx0.1%
5m
DAB incubation (in hood)
18m
Prepare DAB solution (fresh, protect against light) as manufacturer's instruction
Dissolve 1 tablet DAB in 15 ml of Tris-buffered saline, pH 7.6 3,3′-Diaminobenzidine tetrahydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5905
DAB is carcinogenic, so until solution is discarded, all steps should be performed in a protective hood.

Add 12 μl of fresh 30% hydrogen peroxide
Incubate the samples in DAB solution for 8 minutes
8m
Wash with PBS1X
5m
Wash with PBS1X
5m
Mounting
Prepare mounting media (PBS1X: Glycerol 1:1, 0.2% sodium azide). This media lasts up to a year, although it should be discarded if any growths/deposits are observed.

Mount stained samples on glass slides using the mounting media.