Fv3000 confocal system

Neutrophils were stimulated as indicated. In all, 1.0 × 10⁶ cells were seeded on 35 mm confocal dishes (Beyotime), which were coated with 0.001% poly-L-Lysine overnight. Cells were fixed in 4% paraformaldehyde for 30 min at 4 °C. Then the cells were washed with PBS only once, and permeabilized in 0.1% triton X-100 at 4 °C for 10 min. Cells were blocked with 5% BSA at room temperature for 1 h, followed by incubation with anti-PD-L1 antibody (Proteintech, 66248-1-Ig, 1:200) overnight at 4 °C. Cells were washed with PBS only once, and incubated with anti-rabbit IgG (Cell signaling technology, 4412 S, 1:200) for 1 h at room temperature. Cells were washed with PBS and then incubated with DAPI (1 μg/ml) for 15 min at room temperature. Images were captured with FV3000 confocal system (Olympus) and data were analyzed with Imaris (Bitplane, V9.5).

Fresh frozen sections were used to conduct single-molecule fluorescence in situ hybridization by means of RNAScope Multiplex Reagent Kits (ACDBio, USA). The sections were briefly cut to 20 μm thickness and air-dried in a cryostat for less than 20 min. After that, the frozen sections were fixed in ice-cold 4% paraformaldehyde (PFA) for 15 min and dehydrated using an ethanol series. The sections were then treated with H₂O₂ for 10 min and washed with PBS. To initiate hybridization, RNA probes such as VGLUT1, VGAT, and Cre were incubated with the sections in a HybEZ humidified incubator set at 40 °C for 2.5 h. After incubation, the sections were cleaned using ACD wash buffer and then subjected to sequential incubation with AMP1-FL and AMP2-FL reagents for 30 min each, now followed by incubation with AMP3-FL for 15 min. The last step involved staining the sections with diamidino-2-phenylindole (DAPI) to enable visualization of the cell nuclei. The fluorescent signals from the labeled RNA probes and DAPI-stained cell nuclei were observed using an Olympus FV3000 confocal system for image acquisition.

HFL1 cells were grown on coverslips and treated with or without 100 ng/ml His‐PTX3 fusion protein for 24 h. Cells were fixed in 4% paraformaldehyde for 20 min and incubated with antibodies against His (sc‐803, Santa Cruz) and CD44 (GTX83114, GeneTex). For lung sections, samples were incubated with PTX3 (1:100, ab90806; Abcam), fibronectin (1:350 dilution, #15613‐1‐AP, ProteinTech) and α‐SMA (1:100 dilution, GTX100904, GeneTex) at room temperature for 1 h. Samples were incubated with Alexa 568‐ or 488‐conjugated secondary antibodies (Invitrogen) at a 1:200 dilution. The samples were washed with 0.1% Tween 20 in PBS and stained with DAPI (P36935, Invitrogen) on coverslips. The fluorophores were excited by a laser at 405, 488, or 543 nm and observed using an FV‐3000 confocal system (Olympus). Randomly chosen fields were photographed at ×200 magnification under a fluorescence microscope.

BMDMs were isolated from the mice, seeded in the six-well plate (Life Technologies), and the cells were allowed to adhere overnight at 37°C. The cells were stimulated with LPS (100 ng/ml) for 12 h after serum starvation. For inhibitor analysis, the cells were preincubated with DSF (4 μM) or DMSO (0.01%) for 1 h. The confocal images were obtained using an FV3000 confocal system (Olympus), and the data were analyzed with ImageJ (NIH, V2.0.0).

Immunofluorescent labeling was used for gaining the fluorescent green GFP signal and detecting the TRPC4, c-Fos, and BDNF in US-treated and control mouse brains. E18.5 embryos and P3 and P30 mouse pups were sacrificed by decapitation, and whole brains were carefully dissected and immersion fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) overnight at 4°C. Brain samples were then rinsed with PBS and embedded in 4% agarose, and 100-micron-thick free-floating coronal sections were made with a vibratome (Leica, Wetzlar, Germany). Sections were incubated in primary (2 days at 4°C) and secondary (overnight at 4°C) antibody mixtures. All antibodies were diluted in PBS (pH 7.4) supplemented with 0.3 M NaCl and 0.3% Triton X-100. Three 15-min washes in PBS were performed after incubation.
Details of the primary antibodies applied in this study are listed in Table 1. Species-specific secondary antibodies were raised in donkey or goat and conjugated to Alexa Fluor-488, 555, and 647 (Invitrogen, Carlsbad, CA, United States). At the end of the protocol, the sections were incubated with cell nucleus-specific DAPI (Sigma, D9542, 100 ng/ml) for 2 h at RT to help determine layer boundaries. Sections were mounted in Hydromount medium (National Diagnostics Atlanta, GA, United States) and confocal images were obtained with an Olympus FV3000 confocal system.

For confocal microscopy, three 40 μm-thick skin paraffin-embedded specimens from HS-NL and healthy AGR sample groups were assessed, as previously described [10 (link)]. Imaging of the immunostained samples was carried out on an Olympus FV3000 confocal system using a 40× or 60× oil-immersion lens (NAs: 1.40–1.42, respectively). Acquisition settings (laser power, confocal aperture and gain, detector parameters) were identical for all samples. Series of 1 um thick optical sections with 0.5 µm separation in the Z axis were acquired. The overall number of optical sections for each sample was 6. No pixels corresponding to immunostained puncta were saturated. Images for the figures were processed using the Adobe Photoshop CS5 software.
Regularity of the CLDN and DSG1 immunostaining was analyzed by measuring the distance between two adjacent immunostained puncta along the cross section of the cell membrane (defined as a closed line around a DAPI stained nucleus). Inter-puncta distances (120–160 per condition) were measured in the samples. The distances were presented as box plots where the box range represents +/− SD, whiskers indicate the 1.5-fold IQR distance (Q3–Q1) outliers. All datapoints are plotted as grey dots. The mean value is shown as a hollow square within the box.