Eclipse ti s fluorescence microscope

Immunofluorescence assay analysis was carried out by Lipofectamine 2000 transfections of 80–90% confluent 293 T cell monolayers on glass coverslips with co-optimized plasmid concentrations of 1.5 μg of pCAGGS-STAT1-GFP and 1.0 μg of one of the pCAGGS-HA NiV_M P, V, or W expression plasmids with or without the described mutations. Following 18 hours of transfection, cells were exposed to 1,000 U/ml of universal IFN-α (PBL Assay Science, Piscataway, NJ) for 1 hour. Cells were then fixed with 4% (w/v) paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and permeabilized with 1% Triton X-100. Cells were washed, blocked with 3% bovine serum albumin, and incubated overnight with a dilution of 1:1,000 Alexa Fluor 594 conjugated mouse anti-HA (HA.11; Invitrogen). Coverslips with stained cells were mounted onto glass slides and imaged on a Nikon Eclipse Ti-S fluorescence microscope (Nikon Instruments Inc., Melville, NY) using FITC filter (465–495 nm excitation), Texas Red filter (590–650 nm excitation), and X-Cite LEipD light drive (Lumen Dynamics, Ontario, Canada).

Histopathological evaluation of the testicular parenchyma was performed in all patients, and the degree of spermatogenesis was classified according to Johnsen score. Fresh testicular tissues from donors were fixed in 4% paraformaldehyde for 12–24 h at 4 °C, embedded in paraffin, and sectioned. Before staining, tissue sections were dewaxed in xylene, rehydrated using a gradient series of ethanol solutions, and washed in distilled water. Then the sections were stained with PAS/hematoxylin and dehydrated using increasing concentrations of ethanol and xylene. Sections were allowed to dry before applying neutral resin to the coverslips. The staining images were captured with a Nikon Eclipse Ti-S fluorescence microscope (Nikon). Johnsen score of testis sections was identified according to the previous study [17 (link)].

The protocol for immunofluorescent staining was conducted as described above. Primary antibody rat anti-Ki67 monoclonal antibody (1:800, Abcam, Cambridge, UK) to detect cell proliferation and rabbit anti-CD31 polyclonal antibody (1:200, Abcam) to visualize blood vessels. Secondary antibodies goat anti-rat Alexa Fluor 488 (1:200, Invitrogen) and donkey anti-rabbit Alexa Fluor 568 (1:200, Invitrogen) were used for the conjugation of these primary antibodies, respectively. Cell nuclei were stained with DAPI (Invitrogen) for 1 min. Images were captured with a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Melville, NY, USA) and the Image J software . Percentage of Ki67⁺ cells in the stromal compartment and number of blood vessels were counted using the ImageJ software (US National Institutes of Health, USA).

Cells were washed, fixed with 1% paraformaldehyde (PAF) for 20 min, and permeabilized with 0.1% Triton X-100 for 10 min. Cells were blocked with 3% BSA, incubated with the Ab specific for vimentin (1:250; Santa Cruz Biotechnology; mouse anti-human type) and COX-2 (1:100; Cell Signaling; rabbit anti-human type) at 4 °C overnight, and then followed by the anti-mouse 2nd FITC- and anti-rabbit 2nd-rhodamine conjugated Ab (Chemicon) for an additional 1 h at RT. By the end of incubation, DAPI (1:1000; Thermo Fisher Scientific) was added to incubation for cell nucleus staining. After a brief wash, the tissue samples mounted on chamber slides were analyzed under a Nikon Eclipse Ti-S fluorescence microscope (Japan) and photographed using a digital camera. The quantitation of the staining results was performed using the Invitrogen Celleste 5.0 Image Analysis Software (Thermo Fisher). The areas of positive staining for control and CRSwNP patients’ regions of interest (ROI) in the captured images were identified by setting a cutoff value and computed according to the software instructions. The mean intensity of each ROI was calculated as the integrated optical density (OD) of all areas/total area sizes (pixel square) in an ROI. The mean intensity of positive staining was obtained by the above intensity/total ROI number.

The PLA was performed according to the manufacturer’s manual and the following reagents were used: Duolink^® In Situ Detection Reagents Red (Sigma-Aldrich, Darmstadt, Germany; #DUO92008-100RXN), Duolink^® In Situ PLA^® Probe Anti-Mouse PLUS (Sigma-Aldrich, Darmstadt, Germany; #DUO92001-100RXN) and Duolink^® In Situ PLA^® Probe Anti-Rabbit MINUS (Sigma-Aldrich, Darmstadt, Germany; #DUO92005-100RXN). In short, the cells were co-stained over night at 4 °C using the following antibodies: SETD1B (Abcam, Cambridge, UK; #ab113984; 1:1000) and ZEB1 (R&D Systems, Wiesbaden, Germany; #639914; 10 µg/ml). The next day, the PLA probe solution was added to the cells as described in the protocol. After the ligation and amplification steps, the nuclei were stained using ProLong® Gold Antifade reagent (Life Technologies, Darmstadt, Germany). Detection was performed using a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Tokyo, Japan).

The wound healing test was performed to detect the migration rate of HCT-116 and RKO cells after SCU administration. Firstly, the number of cells was counted at RT and the cells were prepared into suspension. Then cell suspension was plated at a density of 5 × 10⁵ cells per well in 6-well plates. Next, a linear scratch was performed with a plastic 10 μl pipette tip on the 6-well plates to make sure that the width of the scratches is as equal as possible. Afterwards, PBS was used to wash the cell fragments from scratches. For HCT-116 cells, the images before drug administration at 0 h as well as after drug administration at 12 h and 36 h were captured via Multifunctional automated inverted Fluorescence Microscopy (Nikon Eclipse Ti-S fluorescence microscope, Nikon). Meanwhile, for RKO cells, the images were captured after drug administration at 0 h, 8 h and 24 h. The transferred rate = (the average distance at 0 h − the average distance at × h)/(the average distance at 0 h) × 100%.