Anti mouse secondary antibody conjugated to alexafluor 488

Cellular CYP3A4 and E-cadherin protein expressions were visually analyzed with immunofluorescence staining and subsequent confocal microscopy. Briefly, on days 1, 7, or 14, cell-laden ICC scaffolds were removed from 24-well plates, washed with PBS, then fixed in 4% paraformaldehyde (PFA) for 5 min. Next, cells were permeabilized with a solution of 0.1% Triton X-100 (Bio-Rad, CA) in PBS for 30 min, washed with PBS, and incubated in 3% BSA blocking buffer for 1 h. Cells were then incubated with mouse monoclonal primary antibodies against CYP3A4 or E-cadherin (Santa Cruz Biotechnology, CA) overnight at 4 °C, washed with PBS, and incubated with anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Life Technologies) for 2 h at room temperature. F-actin was stained with Alexa Fluor 555 phalloidin (Life Technologies). Lastly, cell nuclei were counterstained with 10 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) for 5 min. Fluorescence images were immediately taken using a confocal microscope.

Cells (2 × 10⁴/well) were seeded in 8-well chamber slides (ibidi GmbH). After 24 h recovery, cells were treated with indicated drug concentrations and fixed with 4% paraformaldehyde for 15 min at room temperature and (after washing with PBS) blocked and permeabilized with 5% FCS, 0.3% Triton X-100 in PBS for 1 h. The primary antibody CHOP (Cell Signaling Technology) was added 1:3200 in 1% BSA and 0.3% Triton X-100 in PBS overnight at 4 °C. After washing with PBS, the cells were incubated with anti-mouse secondary antibody conjugated to AlexaFluor488 (Thermo Fisher, 1:500 in 1% BSA and 0.3% Triton X-100 in PBS) for 1 h. Cells were again washed and counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; 1 µg/ml) and wheat germ agglutinin (WGA, 10 µg/ml, Vector Laboratories, CA, USA) in PBS for 10 min. The dyes were removed, and the cells mounted in Vectashield mounting medium (Vector Laboratories, CA, USA) with a coverslip. Images were taken with a Zeiss LSM 700 Olympus (Carl Zeiss AG, Oberkochen, Germany) confocal microscope and CHOP fluorescence intensities per nucleus were measured using ImageJ.

DRG cultures were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized and blocked for immunostaining in TBS-T, 5% skim milk, and 0.3% Triton X-100 for 15 min at room temperature. Immunostaining was performed in TBS-T with 5% skim milk and 0.3% Triton X-100 with mouse anti-β-III tubulin (Millipore; 1:10,000) primary antibody and anti-mouse secondary antibody conjugated to Alexa Fluor 488 (ThermoFisher; 1:5000). Cultures were imaged at 5× magnification using a Zeiss ObserverZ.1 inverted epifluorescence microscope with an automated, motorized stage. Images were stitched automatically with Zen 2 software from Zeiss to produce a master image of all explants on the entire six-well plate. From this master image, quarter-DRG fields were cropped using NIH ImageJ (FIJI build) to create an image set for quantification as recently described in detail by our group (Johnstone et al., 2018 (link)).

Huh-7.5-overexpressing cells were seeded on glass coverslips, fixed with 3% PFA at room temperature for 10 min on the following day, and washed twice with PBS. Subsequently, cells were treated with PBS containing 0.1% Triton X-100 for 10 min at room temperature to permeabilize. After washing twice with PBS, coverslips were incubated in a 5% solution of goat serum in PBS for 1 h at room temperature to block potential nonspecific antibody binding. For CD302 detection, human CD302/CLEC13A mouse monoclonal antibody (R&D Systems) was diluted to 5 μg/mL and incubated at room temperature for 1 h or overnight at 4°C. Afterward, coverslips were washed three times with PBS, followed by an incubation with anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific) in a 1:1,000 dilution at room temperature in the dark for 1 h. After washing again twice with PBS, the nuclei were stained using a 0.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution in water. Finally, after one more washing step with water, mounting of the coverslips in ProLong Gold antifade mountant (Thermo Fisher Scientific) was performed, and they were left to dry overnight at room temperature in the dark. Images were taken with an inverted confocal laser-scanning microscope (Olympus FluoView 1000) with FluoView 1000 imaging software (Olympus, Tokyo, Japan).