Nis elements v4

Samples were imaged on a Nikon N‐STORM system in dSTORM mode which is characterized by a Ti‐E stand with Perfect Focus, 100 × 1.49 NA TIRF objective lens, Agilent Ultra High Power Dual Output Laser bed (170‐mW, 647‐nm laser) for the excitation and Andor IXON Ultra 897 EMCCD camera for the image acquisition. To allow fluorophore blinking, samples were imaged in a PBS based buffer consisting of enzyme solution (catalase 1 µg/mL, Tris [2‐carboxyelthyl] phosphine hydrochloride 4 mmol/L, glycerol 50%, KCl 25 mmol/L, pH 7.5 Tris‐HCl 20 mmol/L, glucose oxidase 50 µg/mL), glucose solution (glucose 100 mg/mL, glycerin 10%), and reducing agent solution (100 mmol/L MEA). For single color (Alexa647) the N‐STORM emission cube was used and the 405 laser power was then increased by 5% every 30 seconds during imaging to reactivate the fluorophore from the dark state. 20 000 frames were captured using Nikon NIS Elements v4.5 with an exposure time of 20 ms, gain 300, and conversion gain 3, and reconstructed using STORM analysis module 3.2, applying the drift correction and the Gaussian rendering. Five separate fields of view (FOV) from three independent experiments were imaged for both mouse and human platelets. Identified points, which represent individual fluorescent blinking events, were filtered on photon count and only those with a count >500 were selected for further cluster analysis.

An inverted fluorescence microscope (Nikon Eclipse Ti, Japan) fitted with FITC 480/30 nm ex 535/45 nm em, Texas Red 560/40 nm ex 630/60 nm em, and DAPI 375/28 nm ex 460/60 nm em filters was used to visualise live and preserved cells in multi-well plates under 200, or 400× magnification and were imaged with a monochrome camera (Nikon DS-QiMc) using proprietary software (NIS Elements v4.60).
An upright fluorescence microscope (Nikon Eclipse Ni, Japan) fitted with a Cy5 620/60 nm ex 700/75 nm em filter and a monochrome camera (Nikon DS-Qi2) with proprietary software (Infinity Analyse v6.4.0) under 200, 400, or 1000× magnification was used for more detailed observation of cells and for obtaining cell size measurements.

Immunofluorescence images and DAPI staining images were acquired every 0.2 μm intervals of z-slice using a Zyla 4.2 sCMOS camera (Andor) mounted on a Nikon Eclipse Ti inverted microscope with an objective lens (Nikon; Plan Apo lambda 100×/1.45 NA) with a spinning disk confocal scanner unit (CSU-W1; Yokogawa) controlled with NIS-elements v4.60 (Nikon). The images in the Figures are the maximum intensity projection or sum intensity projection (chromosome spread samples) of the Z-stack generated with Fiji (v1.53)^{70 (link)} and were processed using Fiji (v1.53) and Adobe Photoshop v23.1.0 (Adobe).

The expression of BUB1B, JNK, and c-Jun in CCA tissues was detected by immunohistochemical (IHC) staining. The details of IHC staining have been described in our previous article^{25 (link)}. The images were acquired and quantified by light microscopy and NIS-Elements v4.0 software (Nikon, Tokyo, Japan).

After 4% paraformaldehyde fixation, the sections were deparaffinized. Antigens of the slides were retrieved by heating for 30 min in citrate buffer, pH 6.0. The slides were labeled with primary antibody in a blocking solution (1:100 dilution) at 4°C overnight, following by counterstained with hematoxylin. Light microscopy (Nikon, Tokyo, Japan) was used to acquire the images, and NIS-Elements v4.0 software was used to quantify the staining (Nikon).

The expression of SOCS3 in CCA tissues were detected by immunohistochemical staining (IHC). The details of IHC staining have been described in our previous article 17 (link). The images were acquired and quantified by light microscopy and NIS-Elements v4.0 software (Nikon, Tokyo, Japan).