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

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:

• 100µL 30% H2O2 ₂O₂
• 9900µL dH2O ₂O

Prepare Normal Goat Serum Blocking Solution in PBS .

To 10mL PBS add:

• 150µL Normal 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 :

• 1µL rabbit anti-GFP (Thermo Fisher, Molecular Probes Cat# A11122, RRID:AB_221569)
• 9999µL PBS .

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.

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).