Feb 17, 2024

DAB Staining for GFP on Free-floating Fixed NHP Brain Tissue

 Forked from Standard DAB Staining for Free-floating Fixed NHP Brain Tissue
DOI
https://dx.doi.org/10.17504/protocols.io.eq2lyjy4mlx9/v1
  • 1Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA
Andreea Bostan
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lyjy4mlx9/v1
Protocol Citation: Aydin Alikaya, William Stauffer 2024. DAB Staining for GFP on Free-floating Fixed NHP Brain Tissue . protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjy4mlx9/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: February 08, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94871
Keywords: ASAPCRN, Immunostaining, DAB, NHP Brain Tissue, fixed nhp brain tissue, nhp brain tissue, green fluorescence protein, brain tissue, fixed brain tissue section, brain tissue section, biotin abc complex, tissue, avidin, gfp
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 to detect Green Fluorescence Protein (GFP).

This protocol has been tested with free-floating non-human primate (NHP) brain tissue that has been fixed with 4% paraformaldehyde, cryoprotected with sucrose gradients, and cryo-sectioned 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 staining net.
Materials
Tissue:
NHP brain tissue sections (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 (30%)
  • Distilled water: dH2O
  • Primary Antibody: Thermo Fisher, Molecular Probes Cat# A11122, RRID: AB_221569
  • Vectastain ABC-HRP Kit, Peroxidase (Rabbit IgG) (PK-4001, Vector Laboratories)
  • Peroxidase (HRP) with Nickel (3,3'-diaminobenzidine) (SK-4100) (Vector Laboratories)







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)
3h
Bring tissue to Room temperature in Phosphate Buffered Saline (PBS, pH 7.2-7.4) on an orbital shaker for 30 minutes. 00:30:00

30m
Prepare Peroxide Solution (0.3 % H2O2) in dH2O.
For 10 mL 0.3% H2O2 use:
  • 100 µL 30% H2O2
  • 9900 µL dH2O


5m
Prepare Normal Goat Serum Blocking Solution in PBS.
To 10 mL PBS add:
  • 150 µL Normal Goat Serum (= 3 drops of serum from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001)

5m
Prepare Primary Antibody Solution (rabbit anti-GFP) at 1:10000 dilution in PBS:
  • 1 µL rabbit anti-GFP (Thermo Fisher, Molecular Probes Cat# A11122, RRID:AB_221569)
  • 9999 µL PBS.
5m
Rinse in PBS on a shaker at Room temperature : 3 x 5 minutes. 00:05:00

15m
Quench endogenous peroxide in Peroxide Solution (0.3 H2O2) on a shaker at Room temperature : 30 minutes. 00:30:00

30m
Rinse in PBS on a shaker at Room temperature : 3 x 5 minutes. 00:05:00

15m
Incubate in Normal Goat Serum Blocking Solution on a shaker at RT: 1 hour. 01:00:00

DO NOT RINSE after blocking serum.
1h
Incubate in Primary Antibody Solution on a shaker at 4 °C Overnight .

12h
Part II (Day 2)
4h
Bring tissue (in the Primary Antibody Solution) to Room temperature on a shaker (30 minutes). 00:30:00

30m
Prepare ABC Solution in PBS (at least 30 minutes before use). 00:30:00
To 10 mL PBS add:
  • 2 drops A from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001.
  • 2 drops B from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001.
5m
Prepare Secondary Antibody Solution (1:200) in PBS.
To 10 mL PBS add:
  • 150 µL (= 3 drops from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001) of Normal Goat Serum.
  • 50 µL (= 1 drop from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001) biotinylated goat anti-rabbit IgG secondary antibody.


5m
Rinse in PBS on a shaker at Room temperature : 3 x 5 minutes. 00:05:00
15m
Incubate in Secondary Antibody Solution on a shaker at Room temperature : 30 minutes. 00:30:00

30m
Rinse in PBS on a shaker at Room temperature : 3 x 5 minutes. 00:05:00
15m
Incubate in ABC Solution on a shaker at Room temperature : 60 minutes. 01:00:00 .

1h
Rinse in PBS on a shaker at Room temperature : 3 x 5 minutes. 00:05:00
15m
Prepare DAB 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 DAB 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 PBS on a shaker at Room temperature : 3 x 5 minutes. 00:05:00

15m
Mount tissue on charged glass slides in 1:8 PBS 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).
Protocol references