Alexa fluor 647 conjugated goat anti mouse igg antibody

Anti-β-actin (catalog no. 4970) was purchased from Cell Signaling Technology (MA, USA). Mouse monoclonal anti-Rho 1D4 antibody was purified from hybridoma cells^{74 (link)–76 (link)}. Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (catalog no. A21236) was from Invitrogen (CA, USA). DAPI fluorescent dye (catalog no. 62248) was purchased from Thermo Fisher (MA, USA). All detergents were purchased from Anatrace (OH, USA). Hydroxylamine hydrochloride (catalog no. 159417) was purchased from Sigma-Aldrich (MI, USA). 9-cis-retinal and 11-cis-retinal were produced by photoisomerization of all-trans-retinal using 420 nm light and purified by normal-phase HPLC using a Phenomenex Luna silica column (catalog no. 00G-4091-P0-AX) and an isocratic gradient of 10% ethyl acetate in hexanes^{77 (link),78 (link)}.

WT-bOpsin-expressing or P23H-bOpsin-expressing HEK293S cells were seeded at 1,000 cells per well in a 24- well plate and incubated at 37 °C with 5% CO₂ overnight. Cells were infected with Nb2-pMXsIG- or Nb16- pMXsIG-viral preparations in the presence of 10 mg mL⁻¹ of polybrene for 2 days. Cells were then fixed with 4% paraformaldehyde/PBS at 400 µl per well for 20 min at RT, followed by permeabilization with 0.01% Triton X-100 for 15 min. After blocking with 10% goat serum for 1 h, cells were incubated with anti-Rho 1D4 antibody overnight at 4 °C. Opsin immunostaining was visualized by incubating cells with 5 µg mL⁻¹ Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen, Catalog no. A212336) for 1 h. Three washes with PBS were performed between each step of incubation with primary and secondary antibodies. Fluorescent images were captured in 24-bit depth with a Keyence BZ-X800 All-in-One Fluorescence Microscope (Keyence, Osaka, Japan) using 4X Nikon Plan Fluor lens and a BZ-X Cy5 filter (#OP-87766). Imaging settings were as follows: excitation 620/60 nm, emission 700/75 nm, dichroic mirror wavelength 660 nm. Ten fields were taken of each well to generate cell images from three biologically independent replicates. Raw images are available in the Source Data file.