Prolong gold containing 4 6 diamidino 2 phenylindole dapi

Briefly, after permeabilization in a solution containing PBS, 0.1% Triton X-100 and 1% BSA (Sigma-Aldrich), cells were incubated with a primary antibody anti-GFP raised in goat (1:500, Rockland, Houston, TX, USA), followed by an incubation with a secondary antibody conjugated to AlexaFluor® 555 (1:1000, Invitrogen, Carlsbad, CA, USA). Slides were mounted with Prolong Gold containing 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). The number of GFP-positive colonies was counted by epifluorescence microscopy and expressed as transduction units (TU) per mL. Knee joint sections with 10 μm thickness were obtained in a cryostat and mounted onto Superfrost slides (Thermo Fisher Scientific). After permeabilization, the sections were incubated with a primary antibody anti-GFP raised in rabbit (1:1000; Invitrogen) overnight at 4 °C, and then processed as described above. Images were acquired using a Zeiss LSM-710 confocal microscope (Carl Zeiss MicroImaging, GmbH, Jena, Germany).

Cells were seeded onto coverslips, fixed using 4% paraformaldehyde (PFA), then permeabilized with 0.5% Triton X-100 for 15 min at room temperature (RT). Cells were incubated overnight at 4 °C with primary mouse antibody against HA (H9658, Sigma-Aldrich, Oakville, ON, Canada, 1:1000) and then with secondary Alexa 488 antibody against mouse (Invitrogen, Life Technologies, Burlington, ON, Canada, 1:1000). The coverslips were mounted using ProLong Gold containing 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen). Cell images were taken with an Olympus BX51 microscope at 40× magnification and Image-Pro Plus (v5.0) software (Media Cybernetics, Inc., Bethesda, MD, USA).
Mean nuclear intensities for immunofluorescence images of each BioID2 construct were determined using ImageJ (v2.1.0). For Regions of Interest (ROI), area and mean pixel intensities (scored between 0 and 255) were acquired by overlaying the binary DAPI image outline mask onto the HA fluorescence image and then averaging the mean ROI intensities and subtracting the background signal acquired from the HEK293 negative control cell line.