Rabbit anti gfp igg

Slides were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol. Antigen retrieval was performed by using IHC-Tek™ Epitope Retrieval Solution (IHC World (Ellicott City, MD, USA), IW-1100-1L) in a steamer. Autofluorescence was blocked by using Image-iT FX Signal Enhancer (Invitrogen, Thermo Fisher Scientific, (Waltham, MA, USA), cat. no. I36933). The protein was blocked by using Normal Goat Serum, 2.5% (Vector Laboratories (Newark, CA, USA), S-1012), and then incubated overnight with primary antibody (rabbit anti-GFP IgG 1:1000 (Abcam (Cambridge, MA, USA), ab5665)) diluted in Antibody Diluent (Dako (Santa Clara, CA, USA), S0809). Slides were incubated the next day in diluent with goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555 (Invitrogen, A32732) at 1:100. Autofluorescence was quenched by using Vector TrueVIEW (Vector Laboratories, cat. no. SP-8400), and nuclei were counterstained with antifade mounting medium with DAPI Hardset (Vector Laboratories, H-1200).

Sections were rinsed in PBS, blocked in 2% normal donkey serum (NDS) for 30 min at room temperature, and incubated on a shaker in rabbit anti-GFP IgG (1:500; Abcam #ab6556) with 2% NDS and 0.3% TX in PBS for 2–3 days at 4°C. They were rinsed in PBS three times (10 min each) and incubated in AlexaFluor 488-conjugated donkey anti-rabbit IgG (1:1000; ThermoFisher #A21206) with 2% NDS for 90 min at room temperature. The sections were rinsed in PBS three times (10 min each), mounted onto gelatin/chromium-subbed glass slides, allowed to air-dry, and coverslipped with ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific, #P36931).