DAB Staining for GFP on Free-floating Fixed NHP Brain Tissue
Aydin Alikaya, William Stauffer
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 .
Before start
Steps
Part I (Day 1)
Bring tissue to Room temperature in Phosphate Buffered Saline ( PBS , pH 7.2-7.4) on an orbital shaker for 30 minutes. 0h 30m 0s
Prepare Peroxide Solution (0.3 % H2O2) 2O2) in dH2O 2O.
For 10mL 0.3% H2O2 2O2use:
100µL30% H2O2 2O29900µLdH2O 2O
Prepare Normal Goat Serum Blocking Solution in PBS .
To 10mL PBS add:
150µLNormal Goat Serum (= 3 drops of serum from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001)
Prepare Primary Antibody Solution (rabbit anti-GFP) at 1:10000 dilution in PBS :
1µLrabbit anti-GFP (Thermo Fisher, Molecular Probes Cat# A11122, RRID:AB_221569)9999µLPBS .
Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 0h 5m 0s
Quench endogenous peroxide in Peroxide Solution (0.3 H2O2) 2O2) on a shaker at Room temperature: 30 minutes. 0h 30m 0s
Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 0h 5m 0s
Incubate in Normal Goat Serum Blocking Solution on a shaker at RT: 1 hour . 1h 0m 0s
DO NOT RINSE after blocking serum.
Incubate in Primary Antibody Solution on a shaker at 4°C . 0h 5m 0s.
Part II (Day 2)
Bring tissue (in the Primary Antibody Solution ) to Room temperature on a shaker ( 30 minutes ). 0h 30m 0s
Prepare ABC Solution in PBS ( at least 30 minutes before use). 0h 30m 0s
To 10mL 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.
Prepare Secondary Antibody Solution ( 1:200 ) in PBS.
To 10mL PBS add:
150µL(= 3 drops from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001) of Normal Goat Serum .50µL(= 1 drop from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001) biotinylated goat anti-rabbit IgG secondary antibody .
Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 0h 5m 0s
Incubate in Secondary Antibody Solution on a shaker at Room temperature : 30 minutes. 0h 30m 0s
Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 0h 5m 0s
Incubate in ABC Solution on a shaker at Room temperature : 60 minutes. 1h 0m 0s.
Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 0h 5m 0s
Prepare DAB Peroxide Substrate Solution in dH2O 2O.
To use the Vector Labs DAB Peroxidase Substrate Kit (SK-4100):
In 5mL dH2O 2O:
- 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.
Incubate in DAB Peroxide Substrate Solution on a shaker at Room temperature:
- 0h 3m 0s -0h 6m 0s.
Note: Watch the tissue closely to avoid high background staining.
Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 0h 5m 0s
Mount tissue on charged glass slides in 1:8 PBS in dH2O 2O and let air dry.
Rinse slides with dH2O 2O and let air dry (preferably in a hood).
Coverslip clean and dry slides with Cytoseal 60 (Thermo Fisher #830-16).
