Sp8 confocal fluorescence microscope

Cells (as specified in each experiment) were seeded on fibronectin-coated coverslips in 24 well plates (2 × 10⁴ cells/well) and cultured overnight. The cells were then fixed with 4% PFA, washed 3 times with PBS and immersed in 0.1% Triton X-100 in PBS for 10 min at room temperature. After washing 3 times with PBS, the cells were incubated with rabbit anti-IGF-1R (Abcam, ab182408), guinea pig anti-Keratin 5 (PROGEN, GP-CK5), or rabbit anti-Ki67 (Cell Signaling, 12202) Abs at 4°C overnight. At the end of incubation, the cells were washed 3 times with PBS and incubated with the corresponding secondary Abs (Alexa Flours, Invitrogen) at 1:500 dilution at room temperature for 1 h. The coverslips were then washed 3 times with PBS for 2 min and mounted with Duolink in situ mounting medium containing DAPI. Images were acquired at 21°C using an SP8 confocal fluorescence microscope (20x dry objective 0.7 NA, 40x dry objective 0.85 NA or 63× oil objective 1.4 NA; Leica) with Leica X version 1.1.0.12420 image software. Skin tissues embedded in OCT were sectioned in 10 µm thickness. The frozen sections were analyzed by immunofluorescence staining as described above. The numbers of Ki67 positive cells or the fluorescence intensities of IGF-1R from five microscopic fields in each section were analyzed and quantified using Image J.

GBM39 cells were plated onto 35 mm, 4‐chamber glass‐bottom dishes at a density of 50 000 cells/well and allowed to adhere at 37°C overnight. Cells were incubated in normoxic (20% O₂) or mild hypoxic (2.0% O₂) conditions for 24 hours prior to treatment with NIR fluorescent smart probe. NO₂‐Rosol (10 μM) was added to the cells and incubated for 20 minutes. Prior to imaging. No washing step was performed. The cells were imaged using a Leica SP8 confocal fluorescence microscope. A 100X (NA = 1.40 OIL) objective lens was used. λ_ex = 550 nm for NO₂‐Rosol. NO₂‐Rosol emission was collected between 680 to 900 nm. Pearson's coefficient was calculated using the Fiji (ImageJ) plugin Coloc 2.

Cochlear specimens after PBS washes underwent staining with 5 μM Caspase 3/7 Green Detection Reagent (Life Technologies, C10723) away from light for 10 min at 37°C. The Leica SP8 confocal fluorescence microscope was utilized for data analysis.

Cochleae or HEI-OC1 cells from different groups were washed in pre-warmed PBS and stained with 5 μM Caspase 3/7 Green Detection Reagent (Molecular Probes, Invitrogen, UK, C10723) in the dark for 10 min at 37°C. Fluorescent images were taken with a Leica SP8 confocal fluorescence microscope.

Immunofluorescence staining was performed as previously described [13 (link)]. All stained samples were mounted and then imaged by a Leica SP8 confocal fluorescence microscope. Whole-slide images of mounted tissue sections were photographed by a GE Amersham Typhoon imager. The in cell western (ICW) assay was performed in multichamber slides (NUNC #154526) with a modified protocol based on the procedures of Egorina et al. [28 (link)] and photographed by a GE Amersham Typhoon imager.

Transfected HEK293 cells were examined with an SP8 confocal fluorescence microscope using 63 × 1.4 NA objective, Leica (Wetzlar, Germany). Alternatively, cells were observed microscopically using an upright Olympus BX51WI equipped with 100 × 1 NA objective and images were captured with a Grasshopper3 CMOS camera (FLIR, Richmond, BC, Canada) controlled by manufacturer provided software. To generate images for quantitative analysis, we kept camera, microscope, and excitation light source (X-Cite 120LEDBoost, Excelitas Technologies, 2% intensity) settings constant. To observe cells with low-level fluorescence, excitation light was set at 15% of intensity. Fluorescence intensities were obtained using Fiji^{37 (link)}. Regions of interest were used to obtain average intensity values from the transfected HEK293 cells (I_cell) and an area without cells (I_background) within the same image. Fluorescence intensity of transfected HEK293 cells (I_norm) for each cell was normalized (I_norm = I_cell − I_background). Each data sample represent average normalized intensity of the transfected cells within an image. We captured 3 to 6 fields of view per dish, using two dishes per construct in every experiment.