Huntingtin Protein Immunostaining and Imaging

Cells grown on No. 1.5 coverslips coated with poly-L-lysine were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at 37 °C. Cells were then permeabilized with 0.25% Triton X-100 in PBS for 10 min. After fixation and permeabilization, cells were immunostained as we described previously [25 (link), 26 (link)]. Rabbit monoclonal anti-huntingtin (1:500, D7F7, Cat #5656S, Cell Signaling) was used. Immunofluorescence was detected using a laser confocal microscope (Nikon A1R). DAPI (4′,6-diamidino-2-phenylindole) was used as a nuclear counterstain. Images were taken with a 60X oil objective (CFI Plan Apochromat Lambda 60X Oil, numerical aperture (NA) = 1.4) under a Nikon A1R laser scanning confocal microscope with a Galvano scanner. DAPI signals were imaged using a 405 nm diode laser at 1% power and detected with a photomultiplier tube (PMT). Htt signals were imaged using a 514 nm diode laser at 2.6% power and detected with a GaAsP detector. NIS-elements acquisition software was used to acquire the images with a resolution of 1024 × 1024 pixels.
A Nikon Eclipse Ts2R microscope equipped with a DS-Fi3 was used to obtain brightfield images from WT, B7 and C12 cells with a 20X objective (S Plan Fluor ELWD 20X/0.45). NIS-elements acquisition software was used to acquire the images with a resolution of 1024 × 1024 pixels.