Feb 07, 2024
Version 2
Standard DAB Staining for Free-floating Fixed NHP Brain Tissue V.2
- 1Department of Neurobiology, University of Pittsburgh
Protocol Citation: Andreea Bostan 2024. Standard DAB Staining for Free-floating Fixed NHP Brain Tissue . protocols.io https://dx.doi.org/10.17504/protocols.io.261ged767v47/v2Version created by Andreea Bostan
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 07, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94869
Keywords: ASAPCRN, Immunostaining, DAB, NHP Brain Tissue
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020519
Abstract
This protocol details the procedure for immunohistochemical 3,3’-Diaminobenzidine (DAB) staining of free-floating fixed brain tissue sections using the avidin/biotin ABC complex.
This protocol has been tested with free-floating non-human primate (NHP) and rodent (mouse, rat) brain tissue that has been fixed (10% formalin or 4% paraformaldehyde), cryoprotected (sucrose or glycerol gradients), and cryo-sectioned 20 µm - 50 µm .
Guidelines
When using 6 well tissue culture plates [Falcon, 353046] to react individual sections, you will need 2+ mL solutions for each well plate.
When using circular staining nets [e.g., Brain Research Laboratories #4115] to react multiple series of sections, you will need 50 mL solutions for each.
Materials
Tissue:
Brain tissue sections (20 - 50 μm).
Materials/Equipment:
- Tissue culture plates or circular staining nets
- Orbital shaker
- Fume hood
- Nitrile Gloves
- Glass slides (charged or subbed)
Reagents:
- Phosphate-buffered saline (PBS)
- Hydrogen Peroxide: H2O2 (3% or 30%)
- Distilled water: dH2O
- Primary Antibody
- Secondary Antibody (to match the host of the primary antibody)
- Normal Serum Blocking Solution (e.g., Normal Horse Serum, S-2000-20; Normal Goat Serum, S-1000-20)
- Vectastain Elite ABC Peroxidase Kit (Standard) (PK-6100) (Vector Laboratories)
- ABC-HRP Kit
Examples:
Vectastain ABC-HRP Kit, Peroxidase (Mouse IgG) (PK-4002, Vector Laboratories)
Vectastain ABC-HRP Kit, Peroxidase (Rabbit IgG) (PK-4001, Vector Laboratories)
- DAB Substrate Kit
Examples:
Peroxidase (HRP) with Nickel (3,3'-diaminobenzidine) (SK-4100) (Vector Laboratories)
ImmPACT DAB (SK-4105)
Safety warnings
Use appropriate care when using hydrogen peroxide (reactive, can cause skin/eye damage) and DAB (suspected carcinogen). Collect DAB solution for chemical waste disposal.
Part I (Day 1)
Part I (Day 1)
3h
3h
Bring tissue to Room temperature in buffer (e g., Phosphate buffered saline, PBS) on an orbital shaker for 30 minutes. 00:30:00 .
30m
Prepare Peroxide Solution (0.3 - 3 % H2O2) in dH2O.
E.g., for 10 mL 0.3% H2O2 use:
- 100 µL 30% H2O2
- 9900 µL dH2O
5m
Prepare Blocking Serum Solution (e.g. Normal Horse, Normal Goat Serum) using a serum that matches the host of the secondary antibody (e.g. Normal Horse Serum for a Horse anti-Mouse secondary, Normal Goat Serum for a Goat anti-Rabbit secondary).
E.g., in 10 mL buffer (PBS) add:
- 150 µL normal serum (or 3 drops of normal serum if using an ABC kit, e.g. Vectastain ABC-HRP Kit, Peroxidase Mouse IgG PK-4002, Rabbit IgG PK-4001)
5m
Prepare Primary Antibody Solution at the appropriate dilution in buffer (e.g., 1:1000 in PBS).
5m
Rinse in buffer (e.g. PBS) on a shaker at Room temperature : 3 x 3-5 minutes. 00:03:00 -00:05:00
15m
Quench endogenous peroxide in Peroxide Solution (0.3 - 3 % H2O2) on a shaker atRoom temperature : 30 - 60 minutes. 00:30:00 -01:00:00
1h
Rinse in buffer (e.g. PBS) on a shaker at Room temperature : 3 x 3-5 minutes. 00:03:00 -00:05:00
15m
Incubate in Blocking Serum Solution on a shaker at RT: 1 hour.
DO NOT RINSE after blocking serum.
1h
Incubate in Primary Antibody Solution on a shaker at 4 °C Overnight , or longer (20 - 72 hours depending on the antibody).
20h
Part II (Day 2)
Part II (Day 2)
4h
4h
Bring tissue (in the Primary Antibody Solution) to Room temperature on a shaker (30 - 60 minutes). 00:30:00 -01:00:00
30m
Prepare ABC Solution in buffer (e.g. PBS) (at least 30 minutes before use). 00:30:00 .
5m
Prepare Secondary Antibody Solution (1:200) in buffer (e.g. PBS).
In 10 mL buffer add:
- 150 µL (= 3 drops of normal serum from a Vector Labs kit) of normal serum (matched to the host of your secondary antibody)
- 50 µL (= 1 drop secondary antibody from a Vector Labs kit) of biotinylated secondary antibody (matched to the host of your primary Antibody )
5m
Rinse in buffer (e.g. PBS) on a shaker atRoom temperature : 3 x 3-5 minutes. 00:03:00 -00:05:00 .
15m
Incubate in Secondary Antibody Solution on a shaker at Room temperature : 30 minutes. 00:30:00 .
30m
Rinse in buffer (e.g. PBS) on a shaker at Room temperature : 3 x 3-5 minutes. 00:03:00 -00:05:00 .
15m
Incubate in ABC Solution on a shaker at Room temperature : 60 minutes. 01:00:00 .
1h
Rinse in buffer (e.g. PBS) on a shaker at Room temperature : 3 x 3-5 minutes. 00:03:00 -00:05:00 .
15m
Prepare Peroxide Substrate Solution in dH2O.
To use the Vector Labs DAB Peroxidase Substrate Kit (SK-4100):
In 5 mL dH2O:
- 2 drops Reagent 1
- 4 drops Reagent 2
- 2 drops Reagent 3
- [optional] 2 drops of Reagent 4 (Nickel) if a black reaction product is desired
Note: Mix well before use. Use immediately.
5m
Incubate in Peroxide Substrate Solution on a shaker at Room temperature :
00:03:00 -00:06:00 .
Note: Watch the tissue closely to avoid high background staining.
6m
Rinse in buffer (e.g. PBS) on a shaker at Room temperature : 3 x 3-5 minutes. 00:03:00 -00:05:00 .
15m
Mount tissue on glass slides (subbed or charged) in 1:8 buffer in dH2O and let air dry.
Rinse slides with dH2O and let air dry (preferably in a hood).
Coverslip clean and dry slides with Cytoseal 60 (Thermo Fisher #830-16).