Rabbit polyclonal anti rfp

For immunohistochemistry (IHC) of vector transfected neurons, a first antibody to the DsRed2 or tdTomato, red fluorescent protein (RFP) was used with subsequent enhancement through diaminobenzidine (DAB) staining. All procedures below were performed at room temperature, unless stated otherwise. Every 24th section from anterior to posterior was incubated in a blocking solution of 10% normal goat serum (NGS) in PBS with 0.3% of Triton X-100 (PBS-T). Sections were then washed with PBS-T and incubated in 1:5000 rabbit polyclonal anti-RFP (Rockland Immunochemicals; Gilberstville, PA USA) in 2% NGS/PBS-T solution overnight at 4°C. On the next day, sections were washed in PBS-T and incubated in 1:200 biotinylated goat anti-rabbit IgG (Vector Laboratories; CA, US) in 2% NGS/PBS-T for 2 h. Afterwards, sections were incubated with 1:200 vectastain elite ABC kit (Avidin-Biotin Complex—Vector Laboratories: CA, USA) in PBS-T for 1 h and visualized with DAB containing 1% nickel ammonium sulfate (0.01% DAB, 1.0% nickel ammonium sulfate, 0.0003% H₂O₂) in tris-buffered saline. To enable comparison of the staining strength throughout the series of whole-brain sections of each monkey, these reaction conditions were carefully controlled to be consistent. The sections were counterstained with 0.1% Neutral Red (Merck 101 369, Frankfurt, Germany).

For immunohistochemistry, sections were rehydrated for 3 × 15 min in PBS-0.5% Triton-X100 or PBS-0.1% Tween-20 (PBS-T) at room temperature (RT) and, then, blocked for 1 h in 1% normal goat serum and 1% normal donkey serum in PBS-T at RT. Sections were incubated with the primary antibody (prepared in blocking solution), overnight at 4°C. The following primary antibodies (1:1,000 µl) were used: rabbit polyclonal anti-RFP (#600-401-379S, Rockland); Living Colors DsRed Polyclonal Antibody (#632496, Takara); chicken anti-GFP (#ab13970, Abcam). Sections were washed for 3 × 15 min with PBS-T at RT and then incubated with the appropriate secondary antibodies (prepared in PBS-T) for 1 h at RT. The used secondary antibodies were: Cy3 AffiniPure Goat Anti-Rabbit IgG (H + L) (1:300; #111-165-003, Jackson ImmunoResearch); Alexa Fluor 488 anti-chicken (1:500; #A11039, Invitrogen); Alexa Fluor 568 anti-rabbit (1:200; #A10042, Invitrogen). Finally, slides were washed 3 × 15 min with PBS-T and either counterstained with NeuroTrace (1:200, 435/455 Blue Fluorescent Nissl Stain (#N21479) or 640/660 Deep-Red Fluorescent Nissl Stain (#N21483), ThermoFisher Scientific) and mounted in Prolong Diamond mounting media (#P3691, ThermoFisher Scientific) or mounted directly in Vectashield Plus Antifade mounting medium with DAPI (#H-2000, Vector Laboratories).