Alexa fluor 568 conjugated goat anti chicken

Cells grown in fluorodishes were fixed with 3% wt/vol paraformaldehyde/phosphate-buffered saline (PBS) for 10 m and permeabilized by 0.4% Triton X-100 in PBS for 15 m. Cell were labeled with chicken anti-BioID2 (1:5,000)^{39 (link)} and 1:1,000 mouse anti-CoxIV (1:1,000, Abcam ab33985). Primary antibodies were detected using 1:1,000 Alexa Fluor 568-conjugated goat anti-chicken (1:1,000, ThermoFisher Scientific A11041) and Alexa Fluor 647-conjugated goat anti-mouse (1:1,000, ThermoFisher Scientific A11029) while Alexa Fluor 488-conjugated streptavidin (1:1,000, ThermoFisher Scientific S32354) was used to detect biotinylated proteins. DNA was visualized with Dapi. Confocal microscopy was performed using a Nikon A1 confocal microscope (60X/1.49 oil APO TIRF Nikon objective) with a charge-coupled CoolSnap HQ camera (Photometrics) linked to a workstation running NIS-Elements software (Nikon, Melville NY).

Cells grown on 1.5 mm glass coverslips were fixed with 3% (w/v) paraformaldehyde/phosphate-buffered saline for 10 min and permeabilized with 0.4% (w/v) Triton X −100/PBS for 15 min. To detect BioID2 fusion proteins, chicken anti-BioID2 (1:5000; BID2-CP-100; BioFront Technologies) was used (18 (link)). The anti-BioID2 antibody was detected using Alexa Fluor 568–conjugated goat anti-chicken (1:1000; A11041; Thermo Fisher Scientific). Alexa Fluor 488–conjugated streptavidin (1:1000; S32354; Thermo Fisher Scientific) was used to detect biotinylated proteins. DNA was detected with Hoechst dye 33342. Coverslips were mounted using 10% (wt/vol) Mowiol 4 to 88 (Polysciences). Epifluorescence images were obtained using a Nikon Eclipse NiE microscope (40 × /0.75 Plan Apo Nikon objective) with a charge-coupled device camera (CoolSnap HQ; Photometrics) linked to a workstation running NIS-Elements software (Nikon). All images were processed in Adobe Photoshop CC 2023 (https://www.techspot.com/downloads/6043-adobe-creative-cloud-photoshop.html) (Adobe) for cropping and brightness/contrast adjustment when applicable.