Leica application suite x las x software

For the evaluation of PD‐L1 on CTCs of NSCLC patients, a semi‐quantitative analysis of PD‐L1 expression was performed, using three NSCLC cell lines (H1975, H460, and A549) expressing different levels of PD‐L1 protein as determined by western blot and triple immunofluorescence analysis (Figs S1 and S3B). The rabbit monoclonal antibody E1L3N was used to detect endogenous levels of total PD‐L1 protein. PD‐L1 expression was considered positive when localized in the cellular membrane, the cytoplasmic component, or both.
The Mean Fluorescence Intensity per pixel (MFI) of PD‐L1 expression for each cell was measured using the Leica Application Suite X (lasx) software (Leica Microsystems). The intensity of PD‐L1 protein was measured among PD‐L1 expressing cells detected in the positive control of H1975, H460, and A549 cells (Fig. S3B). Based on the mean intensity of the selected cell lines, we characterized CTCs as PD‐L1 high, medium, low, and negative, Fig. S5.

For determination of the extracellular pH up to 15 µm from the spheroid surface (pHe), the cell-impermeant pH indicator, carboxy SNARF®-5F dye (S23922, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used. For calibration data, RPMI medium alone or supplemented with either LA or NaHCO₃ was prepared. pH values in these media were determined by HydroDish® HD24 plates (PreSens, Regensburg, Germany) in 37 °C, 5% CO₂. Then, the SNARF-5F dye was added into calibration media (22 µM final concentration). Fluorescence emissions of this probe at 520–580 and 630–700 nm were simultaneously recorded in two channels of the confocal microscope in response to 488-nm laser excitation. Ratios of these fluorescence signals were calculated for each pH and plotted against the respective pH. All fluorescence measurements were performed at 37°C, 5% CO₂. Fluorescence images of the samples were acquired using Leica Application Suite X (LAS X) software (Leica Microsystems, Wetzlar, Germany). For pHe determination, the spheroids were exposed to the cultivation media with or without LA (pH 6.6) or NaHCO₃ (pH 8.1) for three days. Then, the spheroids were loaded with SNARF®-5F dye (22 µM final concentration) for 4 h, and emitted fluorescence was measured. To eliminate autofluorescence, the spheroid without SNARF-5F loading was used as a control.

A randomly chosen series was used to calculate the total volume of the DG in each animal using Nissl staining. Slices were mounted on 2% gelatine-coated glass slides and air-dried at rt for 48 h. The slides were sequentially immersed in the following solutions: 6 min in toluidine blue, 10 sec in bi-distilled water, 2 min in EtOH 70°, 2 min in EtOH 96°, 2 min in EtOH 100°, 2 min in EtOH 100°, and 2 min in Xylene (PanReac, #251769). Next, sections were embedded in DePex (Serva Electrophoresis™, #1824301) and dried for 48 h at rt before imaging. Images were acquired under a THUNDER Imager Tissue microscope equipped with a Leica DFC9000 GTC VSC-09991 camera (Leica Microsystems Ltd., Wetzlar, Germany) and using a 5X dry objective. Images were processed using the Leica Application Suite X (LAS X) software provided by the manufacturer (Leica Microsystems Ltd., Wetzlar, Germany). The DG volume was determined using the freehand selection tool in Fiji software to measure the granule cell layer (GCL) plus the SGZ area on each section of the series. Each area was multiplied by the thickness of the tissue (namely 50 µm) and by the sampling fraction (8). The numbers obtained were summed to calculate the total DG volume, which is expressed in mm³.