N storm

The CDS of CD80/86, CD28, and CD152 were amplified by PCR using specifically designed primers (Table 1). The PCR products were digested and inserted into a pEGFP-N1 plasmid using the EcoR I and BamHI restriction enzymes. The pEGFP-CD80/86, pEGFP-CD28, and pEGFP-CD152 plasmids were transformed into Trans5α chemically competent cells (TransGen), which were subsequently cultured in LB Broth supplemented with 50 mg/L kanamycin for 12 hours at 37°C. The plasmids were then extracted from the cells using an E.Z.N.A. Plasmid Maxi Kit (Omega). HEK293T cells (5 × 10⁵ cells) were seeded into each well of a 12-well plate (VWR) and cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) for 12 hours at 37°C in 5% CO₂. Then, either 1.6 µg of the pEGFP-N1, pEGFP-CD80/86, pEGFP-CD28, or pEGFP-CD152 plasmid was transfected into the HEK293T cells in each well using 4 µL TransIntro™ EL Transfection Reagent (TransGen), according to the manufacturer’s instructions. At 24 hours post-transfection, 4% paraformaldehyde (Biosharp) was added to the cells for 15 minutes to fix them. Afterward, the cells were stained with 100 ng/mL DAPI (Biosharp) for 15 minutes at room temperature. Finally, confocal microscopy (Nikon N-STORM) was used to obtain fluorescence images of the transfected HEK293T cells.

STORM imaging and processing TIRF Imaging experiments were done with a Nikon eclipse Ti2 inverted microscope equipped with Nikon Instruments (N-STORM). A 100 × TIRF objective 1.49NA lens was utilized and imaged using a Hamamatsu C11440 ORCA-flash CMOS 4.0 camera. Images were acquired sequentially 10,000 frames per filter channel at 20 ms time duration. Retinal tissue (N = 3 biological replicates) labeled with JF646 secondary (Additional file 1: Table S2) were excited with 90% laser power from a 647 nm laser and A568 secondary (Additional file 1: Table S2) labeled samples were excited with a 561 nm laser at 100% laser power. Nikon Nd2 files were separated and converted to tiff files per channel by a custom python script. STORM localization analysis was carried out with either the ImageJ thunderstorm plugin (1.3–2014-11–08) or WindSTORM MATLAB code. Data was fitted with a Gaussian PSF model using weighted least-squares estimation for the thunderstorm plugin.

FPALM imaging was performed on a super-resolution microscope NIKON N-STORM equipped with a 100X 1.40 NA Nikon objective lend and an Andor Ixon DU-897E-CS0BV running at approximately 30 Hz (30 ms exposure time). The excitation scheme consisted of an activation laser at 405 nm (Coherent CUBE 405–100 mW) and a readout laser at 488 nm (Coherent Sapphire OPSL 488 nm-50 mW). Specific dichroic mirrors (Chroma, T505LP) and band-pass dichroic filters allowed selection of the emitted signal (Semrock BLP01-488R-25).
The molecules position is found, after background subtraction and thresholding, by means of a gaussian fitting procedure. The rendering of the super-resolution image is obtained plotting the position of each single event as a gaussian spot with standard deviation corresponding to the calculated localization precision. Before the rendering of the final image, a filter on brightness and molecule dimension is applied and unsuitable events are rejected [7] (link).

As previously described (Liu et al., 2021 (link)), isolated mouse islets were plated on 12-well plates overnight and then incubated with or without 10 µM CS for 12 h. Afterwards, islets were incubated in 5 µM Fluo-3 AM working solution (Fluo-3 AM dissolved in anhydrous DMSO was diluted with KRB buffer) at 37°C for 60 min. Subsequently, islets were perfused with KRB buffer-based solutions containing glucose (16.7 mM) or KCl (30 mM) at 37°C at a flow rate of 2 ml/min. Fluorescence imaging was conducted in an N-STORM and A1 confocal scanning microscope (Nikon, Japan).

In the light of a previous delineation, the expression of Collagen-II was observed through ICC [30 (link)]. The treated HNPCs were collected and washed with PBS, and then fixed with 4% paraformaldehyde at room temperature for 15 min. Next, cells were treated with 0.2% of Triton X-100 (P0096, Beyotime, Shanghai, China) for 10 min, and incubated firstly with 10% goat serum (C0265, Beyotime, Shanghai, China) for 20 min, and then with the diluted primary antibody (Collagen II Monoclonal Antibody; MA1-37,493, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 4°C overnight. Subsequently, cells were incubated with the diluted secondary antibody (Rabbit Anti-Mouse IgG H&L; ab6728, Abcam, Cambridge, UK) at room temperature for 1 h. Later, the cells were treated with DAB working solution (P0202, Beyotime, Shanghai, China), and then washed with distilled water. Lastly, the Collagen-II content of cells was observed (under 200 × magnification) using fluorescence microscope (N-STORM, Nikon, Tokyo, Japan).