The ± ENZ-treated cells (VCaP: 100 000 per well; 22Rv1: 40 000 per well) were cultured in charcoal stripped medium on 8-well chamber slides (Ibidi GmbH, #80826). Before staining, the cells were treated with EtOH or 100 nM Dex for 1 h at 37°C. For fluorescent staining, the cells were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 (PB) for 20 min, and washed with PB. The fixed cells were permeabilized for 15 minutes with 0.1% Triton X-100 and 1% BSA, blocked with 1% BSA for 20 min, and incubated o/n at 4°C with anti-GR antibody (1:500). After washing with PB, the cells were incubated for 2 h with Alexa Fluor 647 labeled secondary antibody (1:500), and nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml) (Sigma-Aldrich, #D8417). The fluorescent images were obtained with a Zeiss Axio Observer inverted microscope (40× NA 1.3 oil objective) equipped with a Zeiss LSM 800 confocal module (Carl Zeiss Microimaging GmbH). Image processing was performed using ZEN 2.5 (blue) software (Carl Zeiss Microimaging GmbH), and the images were quantified with ImageJ software (National Institutes of Health).
Lsm800 confocal module
Manufactured by Zeiss
Sourced in Germany
The LSM800 confocal module is a high-resolution imaging system designed for microscopy applications. It provides a core function of generating high-quality, detailed images by scanning a focused laser beam across a sample and collecting the reflected or emitted light. The LSM800 module can be integrated with various microscope platforms to enable advanced imaging capabilities.
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Lab products found in correlation
Axio observer, by Zeiss (4 mentions)
Axio observer inverted microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Zen 2.5 blue software, by Zeiss (1 mentions)
8 well chamber slide, by Ibidi (1 mentions)
Chamber slide, by Ibidi (1 mentions)
Anti eea1, by Cell Signaling Technology (1 mentions)
Texas red anti mouse, by Vector Laboratories (1 mentions)
Anti gm130, by Cell Signaling Technology (1 mentions)
Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
Zen 2.3 blue edition software, by Zeiss (1 mentions)
Phalloidin ifluor, by Abcam (1 mentions)
Alexa fluor 594, by Abcam (1 mentions)
Zen v2.5 blue software, by Zeiss (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Mz10f dissecting microscope, by Leica camera (1 mentions)
Axiovert observer microscope, by Zeiss (1 mentions)
Zen 2.3 blue, by Zeiss (1 mentions)
Lsm780 quasar, by Zeiss (1 mentions)
9 protocols using lsm800 confocal module
1
Glucocorticoid Receptor Immunofluorescence
2
Intracellular Trafficking of Extracellular Vesicles
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Cells were plated in chamber slides (Ibidi GmbH, (Martinsried, Germany) and treated with 109 particles ml–1 of DiIC18-EVs. After 16 h or 24 h of incubation, the cells were fixed with 4% paraformaldehyde, permeabilised with 0.1% Triton X-100 in 1% BSA-PBS and blocked with 1% BSA-PBS. Samples were stained at 4°C overnight with anti-CD44 (1:200, gift from Dr Jalkanen, Turku, Finland), anti-Lamp1 (H4A3, 1:100, IOWA University, Developmental Studies, USA), polyclonal anti-GM130 (1:100) or anti-EEA1 (1:100) (Cell Signaling, Danvers, MA, USA). After washing, the cells were incubated for 1 h at RT with Texas red anti-mouse (1:100) or Texas red anti-rabbit (1:500), (Vector Laboratories, Peterborough, UK)). The nuclei were stained with DAPI (1 μg ml–1, Sigma-Aldrich). Confocal images were obtained with Zeiss Axio Observer with Zeiss LSM 800 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany) using sequential scanning. Co-localisation analyses were conducted using Imaris 7.7.2 software (Bitplane Inc., Belfast, United Kingdom), selecting DiIC18-EVs channel as ROI. Thresholds were automatically adjusted by the software within the ROI. The percentage of ROI material of DiIC18 co-localising with CD44, EEA1, Lamp-1 or GM130 markers was separately analysed from at least eight images containing multiple cells.
3
CLSM Visualization of Synovial Fluid EVs
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The visualization of SF EVs with CLSM was performed as described previously26 (link). In brief, the plasma membranes of EVs were stained with CellMask Deep Red plasma membrane stain (Thermo Fisher Scientific, Waltham, MA, USA) that was soluted together with Alexa Fluor 568-labeled HA binding complex (HABC) to visualize HA27 (link). Phalloidin-iFluor, conjugated to Alexa Fluor 594 (Abcam, Cambridge, UK), was used to validate the visualized structures as actual EVs. CellMask and HABC stainings were first performed on unprocessed SFs (n = 29), and then on the same samples centrifuged at 1000 × g for 10 min to deplete any cell fragments. For phalloidin staining, SF was (ultra)centrifuged (1000 × g for 10 min, 1200 × g for 20 min, < 110,000 × g for 90 min), and the ibidi coverglass the EV fraction was placed on was coated with poly-D-lysine hydrobromide. The Zeiss Axio Observer inverted microscope equipped with the Zeiss LSM 800 confocal module was used for imaging, and the initial image acquisition was carried out using the ZEN 2.3 blue edition software (Carl Zeiss MicroImaging GmbH, Jena, Germany). The area and intensity of the stainings, particle counts, and their size distribution were determined with the ImageJ/Fiji software (NIH, Bethesda, MA) as outlined previously26 (link).
4
Confocal Microscopy for 3D Cell Cultures
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The fluorescent images were obtained with a Zeiss Axio Observer inverted microscope (40 × NA 1.3 oil or 63 × NA 1.4 oil–objectives) equipped with LSM800 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany). For live cell imaging, Zeiss XL-LSM S1 incubator with temperature and CO2 control was utilized. ZEN v2.3 SP1 (black) software (Carl Zeiss Microimaging GmbH) and ImageJ software (National Institute of Health, Bethesda, MD, USA) were utilized for image processing, 3D rendering and image analysis such as measurements of staining intensity, invasion, spheroidal growth and EV secretion in 3D cultures.
5
Confocal Imaging of Spheroid Cultures
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Confocal imaging of spheroid cultures was performed with Zeiss Axio Observer microscope, with 20 × NA 0.8 and 63 × NA 1.4 oil objectives, equipped with LSM800 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany). ZEN v2.5 Blue software (Carl Zeiss Microimaging GmbH) and ImageJ were utilized for image processing and 3D rendering.
6
GFP and DsRed2 Fluorescence Imaging
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Transformed roots expressing GFP were further identified by fluorescence detection using an MZ10F dissecting microscope from Leica (Solms, Germany) equipped with a mercury UV-lamp and an eGFP band-pass or a DsRed2 long-pass filter. Confocal Laser Scanning Microscopy (CLSM) was carried out on an Axiovert Observer microscope with an LSM800 confocal module from Zeiss (Jenna, Germany), using a 488 nm diode laser line for excitation and collecting an emission wavelength range of 515 ± 15 nm, with the pinhole set to 1.0 airy unit and detector settings set to collect the full dynamic range of emitted fluorescence.
7
Confocal Microscopy Imaging Protocol
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The confocal microscope images were obtained with a Zeiss Axio Observer inverted microscope (10x, 40x (oil) or 63x (oil) -objectives) equipped with LSM800 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany). TAMRA-FP, DAPI, and secondary antibodies were imaged with 561 nm (λex 543 nm/λem 567 nm), 405 nm (λex 353 nm/λem 465 nm), and 640 nm (λex 653 nm/λem 668 nm) lasers, respectively. Secondary antibody used with bHABR (Figure S13 ) was imaged with 488 nm (λex 495 nm/λem 519 nm) laser. ZEN 2.3 (Blue) and ZEN 2.3 lite softwares (Carl Zeiss Microimaging GmbH) were utilized for image processing and image analysis.
8
Confocal Microscopy of Cell Dynamics
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Confocal microscopy was carried out on Zeiss Axio Observer inverted microscope with either LSM780-Quasar (34-channel spectral, high-sensitivity gallium arsenide phosphide detectors, GaAsP) or LSM800 confocal module controlled by the Zen software. All live cell imaging was carried out in a temperature/humidity/CO2-regulated chamber. The signaling/cytoskeletal waves on the cell ventral surface were obtained by capturing the confocal slice of the very bottom of the cell.
9
Confocal Imaging of Organoid Microglia
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All confocal images were obtained with a Zeiss Axio Observer inverted microscope (10×, 20×, 40× (oil) or 63× (oil) -objectives) equipped with LSM800 confocal module (Carl Zeiss Microimaging GmbH). DAPI and secondary antibodies were imaged with 405 nm (λex 353 nm/λem 465 nm), 488 nm (λex 495 nm/λem 519 nm) and 561 nm (λex 543 nm/λem 567 nm) lasers, respectively.
For imaging of representative figures, we used Leica Thunder 3D tissue imager, Figure 1b, (Leica microsystems CMS GmbH, Wetzlar, Germany), 10× objective using the Leica X application suite version 3.7.2.22383. The Zeiss confocal with 20× objective for Figure 1c–f, 40× for Figure 1g and 63× for Figures 1i, 2,Figures S1c–e and S2 .
For quantification of morphology, we employed two imaging devices to acquire whole slice Z-stack tiles, capturing the entire Z-dimensional morphology of the microglia. We imaged the day 120 organoids at the Zeiss confocal, with seven focus planes (merged into maximal orthogonal projection), using the EC Plan-Neofluar 10×/0.30 M27 objective. Day 35 and day 66 organoids we imaged using 3D HISTECH Pannoramic 250 Flash III with 20× objective (3DHISTECH Ltd., Budapest, Hungary) and the extended view images exported to TIF for downstream analysis using the Caseviever version 2.4.
For imaging of representative figures, we used Leica Thunder 3D tissue imager, Figure 1b, (Leica microsystems CMS GmbH, Wetzlar, Germany), 10× objective using the Leica X application suite version 3.7.2.22383. The Zeiss confocal with 20× objective for Figure 1c–f, 40× for Figure 1g and 63× for Figures 1i, 2,
For quantification of morphology, we employed two imaging devices to acquire whole slice Z-stack tiles, capturing the entire Z-dimensional morphology of the microglia. We imaged the day 120 organoids at the Zeiss confocal, with seven focus planes (merged into maximal orthogonal projection), using the EC Plan-Neofluar 10×/0.30 M27 objective. Day 35 and day 66 organoids we imaged using 3D HISTECH Pannoramic 250 Flash III with 20× objective (3DHISTECH Ltd., Budapest, Hungary) and the extended view images exported to TIF for downstream analysis using the Caseviever version 2.4.
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