Fluoromount g with dapi

Cells were washed 3 times with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, washed 3 times with PBS, incubated with PBS supplemented with 0.1% saponin and 0.5–1% BSA (blocking buffer) for 30 min at room temperature, incubated with primary antibodies diluted in blocking buffer for 30 min at 37 °C, washed 3 times with PBS, incubated with secondary antibodies diluted in blocking buffer for 30 min at 37 °C, washed twice with PBS and once with distilled water, and mounted on slides using Fluoromount-G with DAPI (Cat# 0100-20, Electron Microscopy Sciences). Alexa Fluor 546-phalloidin was added for 15 min at room temperature, after the secondary antibody was removed and the coverslip was washed 3 times in PBS.

KB human carcinoma cells (ATCC) which overexpress FRs and immortalized human primary dermal fibroblasts (a kind gift from Dr. Howard Worman, Columbia University) which have normal FR expression levels were maintained in DMEM supplemented with 10% FCS at 37 °C. After a brief wash in 37 °C PBS (Lonza), cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Coverslips were then washed three times with PBS for 5 min and incubated with blocking buffer (0.6 mM BSA, 0.1% Tween-20 in PBS) for 30 min. Cells were then immunostained with a rabbit anti-FR alpha polyclonal antibody (Thermofisher Scientific, PA5-42004) at 1:100 ratio in blocking buffer for 1 h at room temperature. Cells were then washed three times with blocking buffer for 5 min before the addition of a goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (Thermofisher Scientific, A-11034) at 1:300 ratio in blocking buffer for 1 h at room temperature. Cells were washed three times with blocking buffer for 5 min and with PBS for 5 min before being mounted in Fluoromount-G with DAPI (Electron Microscopy Sciences) and imaged by wide-field fluorescence imaging on an inverted Nikon Eclipse Ti-E microscope equipped with appropriate optical filters for DAPI and Alexa Fluor 488 detections.

HCT116 cells were grown in 8-wells culture slides and were fixed with 4% paraformaldehyde for 30 min. Slides, after permeabilization with triton X-100 0.1%/PBS for 10 min, were blocked with 3% normal goat serum/PBS for 30 min at room temperature. They were then incubated with anti-CD147 antibody (mouse monoclonal anti-human, dilution, 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C, followed by incubation with mouse secondary antibody (dilution 1:500, Alex Fluor 633 conjugated, clone 1H6, Thermo Fisher Scientific UK Ltd.) for 30 min. Afterwards, slides were incubated with fluorescein isothiocyanate–labeled phalloidin for 45 min to counterstained cytoskeleton. Subsequently, the slides were mounted with DAPI (Fluoromount G with DAPI; Electron Microscopy Sciences, Hatfield, PA, USA). Control samples were incubated with secondary antibody only. Slides were examined using an inverted confocal microscope (Nikon A1 MP, Instruments Inc. Melville, NY, USA). Images were taken using ImageJ software version 1.41 (NIH, Bethesda, Rockville, MD, USA). Analyses were performed using the OrientationJ plugins to analyze the coherency [19 (link)]. Quantification of nuclear and cytoplasm fluorescence signal was performed using the “Intensity Ratio Nuclei Cytoplasm Tool” plugin of Image J (NIH, Bethesda, MA, USA).

To determine the location and depth of recording electrodes, we coated the shanks with 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) (Thermo Fisher #D282, 50 mg/mL solution in ethanol), prior to insertion into the brain. We dipped the electrode tips 5 times into the DiI solution and allowed the coating to dry for 30 s between immersions. Fluorescent electrode tracks were then visualized in coronal sections of fixed brain tissues. Mice were anesthetized with isoflurane and injected intraperitoneally with pentobarbital sodium (150 mg/kg). They were then perfused transcardially with cold (4°C) PBS followed by cold 4% paraformaldehyde. Brains were extracted and fixed in 4% paraformaldehyde for 6–12 hr and then cryoprotected in a 30% (w/v) sucrose solution in PBS until they sank. Brains were sectioned at 50 µm on a freezing microtome (Leica), mounted on glass slides, and coverslipped with mounting media containing DAPI (Fluoromount-G with DAPI, Electron Microscopy Sciences). Slides were imaged using an Olympus slide-scanner (Olympus BX61VS, Japan).