Pancreatic islets and rat insulinoma INS-1E β-cells were embedded in O.C.T. compound (Sakura, Tokyo, Japan) and frozen. Cryostat sections (6 μm thick) were cut, mounted, and blocked in 2% bovine serum albumin (Sigma-Aldrich,) with 0.1% Triton X-100 (Sigma-Aldrich) for 30 min at RT. For the autophagy analyses, sections were probed with polyclonal rabbit anti-LC3 antibody (Novus) and guinea pig polyclonal anti-insulin antibody (Life Technologies, Carlsbad, CA, USA) and then incubated for 1 h at RT with Alexa Fluor® 568 donkey anti-rabbit antibody and Alexa Fluor® 488 goat anti-guinea pig (both from Life Technologies). DAPI nuclear counterstain was applied, and then the sections were washed three times with phosphate buffer saline, mounted with VectaShield mounting medium (Vector Laboratories, Burlingame, CA, USA), and observed under a fluorescent microscope (Olympus FV1200 Confocal system with a motorized stage and SIM scanner). Antibodies and dilutions used are listed in Table 2 .
Fv1200 confocal system
Manufactured by Olympus
Sourced in Japan
The FV1200 confocal system is a high-performance laser scanning microscope designed for advanced imaging applications. It offers high-resolution, real-time imaging with exceptional image quality. The system is equipped with multiple laser lines and detection channels to enable flexible and versatile imaging capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Ix83 inverted microscope, by Olympus (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 488 goat anti guinea pig, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rabbit anti lc3 polyclonal antibody, by Novus Biologicals (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Ix81 inverted microscope, by Olympus (1 mentions)
Multicolor kit, by Absin (1 mentions)
6 protocols using fv1200 confocal system
1
Immunofluorescent Localization of Autophagy Markers in Pancreatic Islets
2
Confocal Imaging of Hydrated Biofilms
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The non-invasive confocal imaging of fully hydrated biofilms was carried out by means of a fixed-stage Ix83 Olympus inverted microscope coupled to an Olympus FV1200 confocal system (Olympus, Shinjuku, Tokyo, Japan). The objective lens was a ×63 water-immersion lens (Olympus). Specimens were stained at room temperature with LIVE/DEAD® BacLightTM Bacterial Viability Kit solution (L7012, Molecular Probes B. V., Leiden, The Netherlands). A staining time of 8 ± 1 min, in a 1:1 fluorochrome ratio was used to obtain the best fluorescence signal at the corresponding wavelengths (Syto9, 515–530 nm and propidium iodide, PI > 600 nm). At least three different and demonstrative locations of the discs were selected for the study. A z-series of scans (xyz) of 1 μm thickness (8 bits, 1024 × 1024 pixels) were analyzed thanks to the configuration of the CLSM control software. Image stacks were analyzed by using the Olympus® software (Olympus®). To quantify the biomass and cell viability within the biofilm, total fluorescent staining of the confocal micrographs was analyzed using an open source image analysis software (Fiji ImageJ) by measuring voxel intensities from two-channel images and, thus, calculating the percentage of the biomass and cell viability within the stacks [25 (link)].
3
Live/Dead Biofilm Imaging via CLSM
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Non-invasive confocal imaging of fully hydrated biofilms was carried out using a fixed-stage Ix83 Olympus inverted microscope coupled to an Olympus FV1200 confocal system (Olympus; Shinjuku, Tokyo, Japan). LIVE/DEAD® BacLight™ Bacterial Viability Kit solution (Molecular Probes B. V., Leiden, The Netherlands) was used to stained the biofilms at room temperature. The fluorochromes were incubated (ratio 1:1) during 9 ± 1 min to obtain the optimum fluorescence signal at the corresponding wave lengths (Syto9: 515–530 nm; Propidium Iodide (PI): > 600 nm. The CLSM software was set to take a z-series of scans (xyz) of 1 μm thickness (8 bits, 1024 × 1024 pixels). Image stacks were analyzed by using the Olympus® software (Olympus). Image analysis and live/dead cell ratio (i.e. the area occupied by living cells divided by the area occupied by dead cells) was performed with Fiji software (ImageJ Version 2.0.0-rc-65 / 1.52b, Open source image processing software).
4
Electromigration of Cell Surface Receptors
5
Multicolor Immunohistochemistry Staining Protocol
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For the mIHC staining in this study, primary antibodies (detailed information showed in supplementary Table 3 ) as well as a multicolor-kit (Absin, China) were used. We used Opal 520 channel for PD-L1 [fluorescein isothiocyanate (FITC), a green fluorescence stain], Opal 570 channel and Opal 670 for HLA-DR [cyanine 3 (Cy3), an orange fluorescence stain for Fig. 6 . cyanine 5 (Cy5), a red fluorescence stain for Fig. 5 .], Opal 670 channel for FEN1 [cyanine 5 (Cy5), a red fluorescence stain], and DAPI (4′,6-diamidino-2-phenylindole, a blue fluorescence stain). All the 20 slides were observed and imaged by Olympus FV1200 confocal system (Tokyo, Japan). All the images were analyzed by ImageJ software (NIH, Bethesda, MD, USA).
6
LIVE/DEAD Biofilm Viability Imaging
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Biofilms grown on HA discs were stained with LIVE/DEAD® BacLightTM Bacterial Viability Kit solution (Molecular Probes B. V., Leiden, The Netherlands) at room temperature, with 1:1 fluorocromes ratio, in the dark for 10 min (SD = 1). The obtained fully hydrated biofilms were studied with a confocal laser scanning microscope, using a fixed-stage Ix83 Olympus inverted microscope, coupled to an Olympus FV1200 confocal system (Olympus; Shinjuku, Tokyo, Japan) with a ×63 water-immersion lenses (Olympus, Shinjuku, Tokyo, Japan). At least three separate and representative locations were selected from the HA discs covered with biofilm. With the use of a dedicated software (Olympus® software (Olympus, Shinjuku, Tokyo, Japan) and Image analysis FIJI® software (Image J v. 2.0.0-rc-65 /1.52b) a z-series of scans (xyz) of 0.5 µm thickness (8 bits, 1024 × 1024 pixels) were analyzed.
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