Feb 15, 2022
Peroxidase/DAB protocol
- 1The Norwegian University of Science and Technology
- Sandvig lab
Protocol Citation: Christiana.bjorkli 2022. Peroxidase/DAB protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.b44fqytn
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: February 15, 2022
Last Modified: February 15, 2022
Protocol Integer ID: 58215
Abstract
Peroxidase/DAB protocol for chromogen labelling
Leave sections in 0.125 Molarity (M) phosphate buffer (PB) at 60 °C
2h
Dilute 0,4M PB. Store in refrigerator for up to 1 week.
100 ml: 31,25 ml0.4 Molarity (M) PB + 68,75 ml H2O
Rinse sections 2 x 10min in PB
20m
Rinse sections 1 x 10 min in 0,5% TBS-Tx
10m
Tris: 3,03 gram/500 mlH2O
NaCl: 4,48 gram/500 mlH2O
Triton-X-100: 2,5 ml/500 mlH2O
Use HCl to adjust the 8 . Store in refrigerator for up to one week.
Incubate with 10% goat serum in TBS-Tx
30m
Incubate with primary antibody (AT8; MAP2) 1:1000 in TBS-Tx with 5% goat serum,overnight in refrigerator 4 °C
12h
Rinse 3 x 10 min in TBS-Tx
Note
Mix secondary antibody during first wash, and start dissolving DAB during second wash. Leave DAB on heated stirrer for 2 hours, then leave on bench.
30m
DAB
Dissolve 1 tablet (10 mg) in 15 ml Tris-HCl by leaving it on a stirrer with heat (max 50 °C ) for about 2 hours. Add 12 µL H2O2 just before use and filtrate.
For dissolving the DAB use a brain cup (or any other cup you can throw away)
For filtrating the DAB use a disposable syringe with filter.
Safety information
Remember that DAB and H2O2 are hazardous chemicals. Wear gloves, work in the hood, and put the DAB-solution and the first HCl-waste in the DAB waste-bottle.
Incubate with secondary antibody, biotinylated goat anti-mouse, 1:500 in TBS-Tx with 5% goat serum, in room temperature
Note
Mix ABC right before next step
1h 30m
From the ABC-kit, put 1 drop of solution A and 1 drop of solution B in 5 mL TBS-Tx. Mix well and leave on the bench for 30 min before use.
Rinse sections 3 x 10 min in TBS-Tx
30m
Incubate with ABC in room temperature
1h 30m
Rinse 3 x 10 min in TBS-Tx
30m
Rinse 2 x 5 min in Tris-HCl
10m
Incubate with DAB
10m
Rinse 2 x 5 min in Tris-HCl
10m
Mount sections in Tris-HCl on Superfrost slides and let them dry
Coverslip with Xylene and Entellan