Aug 23, 2025
Free Floating DAB Staining
- Ashley Harms1,
- Jhodi Webster1
- 1University of Alabama at Birmingham
Protocol Citation: Ashley Harms, Jhodi Webster 2025. Free Floating DAB Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pxqqgwz/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: October 14, 2023
Last Modified: August 23, 2025
Protocol Integer ID: 89279
Keywords: ASAPCRN, immunohistochemical staining, floating tissue section, floating dab, antibody penetration by processing section, permanent chromogenic precipitate for the microscopic visualization, antibody penetration, antigen distribution, analysis of antigen distribution, tissue section, dab, tissue integrity, permanent chromogenic precipitate, microscopic visualization
Abstract
This protocol describes the procedure for performing 3,3'-Diaminobenzidine (DAB) immunohistochemical staining on free-floating tissue sections. The method is optimized to preserve tissue integrity and enhance antibody penetration by processing sections without being mounted to a slide. It results in a stable, permanent chromogenic precipitate for the microscopic visualization and analysis of antigen distribution.
Troubleshooting
Safety warnings
Be sure to do DAB developing in a fume hood and dispose of all apparatus that touched DAB solution in bucket of bleach and water.
DAY 1
Wash sections 3x5min in TBS
Quench sections with solution for 5min at RT
Quenching solution (10mL – 3% H2O2 in 1XTBS):
4.5mL TBS
4.5mL MeOH
1mL 30% H2O2
Wash 2x5min TBS
Put sections in antigen retrieval for 30mins at 37C with agitation
Antigen retrieval in TBS:
10mM sodium citrate
0.05% Tween-20
Wash 3x5min in TBS
Block in 5% serum + TBST for at least 1hr @ room temp
24-well plate (500μL/well): dilute primary Ab in TBST + 1% serum (of secondary host animal).
Incubate overnight at 4C
Day 2
Wash sections 3x10min in TBST
Incubate sections with diluted biotinylated secondary Ab [(1:1000) in TBST + 1% serum] for 2hrs at RT in the dark
Wash sections 3x10min in TBS in the dark
Dilute ABC solution (1:5 in TBS) and incubate sections for 30min at RT (using 2mL/well)
Wash 3x10mins in TBS
Follow DAB kit instructions (work in the hood, set up a bucket with bleach and water)
Prepare substrate working solution. To every 5mL of diH20, add:
2 drops of Buffer Stock Solution
4 drops of DAB Stock Solution
2 drops of Hydrogen Peroxide Solution
If gray-black reaction is desired, add 2 drops of Nickel Solution
Move samples to substrate solutions and develop until sufficient colour change is evident; around 3-6mins
Immediately wash 3x5min in TBS
Mount sections on slides and dry for approximately 30min to 1hr or just until sections are adhered to slide.
Dehydrate:
70% ethanol 2x3min
90% ethanol 2x3min
95% ethanol 2x3min
100% ethanol 2x3min
Citrisolve 2x3min
Using Permount, cover with coverslip and dry several hours up to 2 days @RT.