Neutralization protocol was based on previously reported experiments with the SARS-CoV-2 S pseudotyped lentivirus73 . 293T-ACE2 cells were seeded on tissue-culture-treated, poly-lysine treated 96-well plates at a density of 10,000 cells per well. Cells were allowed to grow overnight at 37 °C, and then treated with selected inhibitors as described above for live virus infection. Lz-Green SARS-CoV-2 S pseudotyped lentivirus was added to 293T-ACE2 cells treated with 5 µg/mL polybrene and incubated for 48 h before imaging. Cells were fixed with 4% PFA for 1 h at room temperature, incubated with DAPI for 10 min at room temperature, and imaged with BZ-X700 all-in-one fluorescent microscope (Keyence). The estimated area of DAPI and GFP fluorescent pixels was calculated with built-in BZ-X software (Keyence). There were five biological replicates for each condition, and the biggest outlier was removed from analysis due to inherent variability in the assay.
Bz x software
Manufactured by Keyence
Sourced in Japan
The BZ-X software is a core component of Keyence's BZ-X series of microscope systems. It provides the essential functionality to operate and control the microscope hardware, enabling users to capture, view, and analyze microscopic images and samples.
Automatically generated - may contain errors
Lab products found in correlation
Bz x710, by Keyence (3 mentions)
Bz x800 microscope, by Keyence (3 mentions)
Bz x700 all in one fluorescent microscope, by Keyence (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Bz x710 microscope, by Keyence (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bz x710 fluorescence microscope, by Keyence (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Las af software, by Leica Microsystems (1 mentions)
Tcs sp5 confocal microscope, by Leica Microsystems (1 mentions)
Fitc conjugated anti b220, by BioLegend (1 mentions)
Anti gl7, by BioLegend (1 mentions)
Fluoromount, by Southern Biotech (1 mentions)
Zen software, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Cell observer, by Zeiss (1 mentions)
Axio imager m1, by Zeiss (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
11 protocols using bz x software
1
SARS-CoV-2 S Pseudotyped Lentivirus Neutralization Assay
2
Calcium Oscillation Dynamics in EBs
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EBs of ES/scLRP6S/scFZD8L were stimulated with 1 μg/ml BSA-FL from day 2 to day 3 and transferred into gelatinized dish on day 6 of differentiation. On day 17, the cells were stained with Fluo-8 AM calcium indicator (AAT Bioquest) according to the manufacturer’s instructions. Time-lapse images of calcium oscillation were acquired using BZ-X710 fluorescence microscope and BZ-X software (Keyence). Signal intensities were quantified using ImageJ software.
3
Immunohistochemical Analysis of Pancreatic Islets
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Pancreatic samples obtained from the edge of the surgically resected portion had been fixed, paraffin-embedded, and archived. The tissues were serially sectioned at a thickness of 4 μm at 2 random levels and processed for histological analysis. All hematoxylin and eosin–stained pancreatic sections were confirmed by in-house pathologists to contain no pancreatic tissues with tumor elements, pancreatitis and/or autolysis, and immunohistochemistry was then performed. Nuclear counterstaining of sections was performed with DAPI (Vector Laboratories). Histochemical reactions were assessed at the same time in all groups studied, using the same lot of antibodies at dilutions and light exposure times predetermined to maximize sensitivity and minimize nonspecific staining. To identify islet dedifferentiated cells, the tissues were stained with a cocktail of 4 major pancreatic hormones (4H: insulin, glucagon, somatostatin, and pancreatic polypeptide) and ChgA according to a previous report (14 (link)). All antibodies used for immunohistochemical analysis are listed in Supplemental Table 4 . Fluorescence images were captured using a BZ-X710 microscope with BZ-X software (Keyence).
4
Immunofluorescence Labeling of Cardiac Cells
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Based on the standard protocol of immunofluorescence labeling as previously described [26 (link), 30 (link)], EBs differentiated from ES/scLRP6S/scFZD8L were fixed and stained with anti-cardiac α-actinin primary antibody (Abcam). Signals were visualized by anti-mouse IgG-Alexa594 secondary antibody (Life Technologies), and nuclear counterstain was performed using 4’,6-diamidino-2-phenylindole (DAPI, Life Technologies). Images were acquired using a BZ-X710 fluorescence microscope and a BZ-X software (Keyence). For BSA-FL detection, EBs differentiated from ES/mock and ES/scLRP6S/scFZD8L on day 3 with vehicle or BSA-FL treatment were fixed and incubated in 10%/20%/30% sucrose/PBS series. Frozen sections were collected from these EBs embedded in OCT compound (Sakura Finetek Japan) and counterstained in DAPI-containing PBS. Confocal images were obtained with TCS SP5 confocal microscope and LAS-AF software (Leica Microsystems).
5
Immunofluorescent Tissue Staining Protocol
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Spleens were frozen in OCT at −80°C before sectioning in a Microm‐HM‐525 cryostat. Tissue sections were fixed in acetone for 5 min and blocked with 3% BSA/PBS. Sections were stained overnight with conjugated antibodies: FITC‐conjugated anti‐B220 or anti‐GL7, and biotinylated anti‐CD4 (all Biolegend) with a secondary stain of AlexaFluor‐594‐conjugated streptavidin (Thermo Life Technologies, MA, USA). Sections were mounted with Fluoromount (Southern Biotech, AL, USA) and imaged with a fluorescence BZ‐X850 microscope with a BZ‐X software (Keyence, Osaka, Japan).
6
High-Resolution Imaging of Barrel Cortex and TRN
7
SARS-CoV-2 Pseudovirus Neutralization Assay
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The neutralization protocol was based on previously reported neutralization methods utilizing SARS-CoV-2 S pseudotyped lentivirus (Crawford et al., 2020 ). 293T-ACE2 cells were seeded poly-lysine treated 96-well plates at a density of 10,000 cells per well. Cells were allowed to grow overnight at 37°C. LzGreen SARS-COV-2 S pseudotyped lentiviruses were mixed with saRBD-1, or VHH52 control antibody. Immunized alpaca serum was used as positive neutralization control, while virus alone was used as negative control. Dilutions of antibodies ranged from 177 nM to 170 pm for saRBD-1 and 26.3nM and 25 pM for Fc-saRBD-1, and 6.57 nM to 4 pM Bi-saRBD-1. Virus-antibody mixture was incubated at 37°C for 1 hour after which polybrene was added up to 5 μg/ml and the mixture was added to 293T-ACE2 cells. Cells were incubated with neutralized virus for 44 hours before imaging. Cells were fixed with 4% PFA for 1 hour at RT. Fixed cells were washed with PBS 2x, then incubated with 10 μg/mL DAPI for 10 minutes at RT, imaged with BZ-X700 all-in-one fluorescent microscope (Keyence). Estimated area of DAPI and GFP fluorescent pixels were calculated with built in BZ-X software (Keyence).
8
Quantitative Analysis of Oligodendrocyte Lineage Cells
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Images were acquired using an epifluorescence microscope (Zeiss Axio-imager M1) or a LSM 510 Meta confocal microscope (Zeiss). Confocal images represent maximum intensity projections of z-stacks, with step sizes of 0.5–3 μm. Whole brain section images were acquired as tiled arrays using an epifluorescence microscope equipped with a computer-controlled stage (Cell Observer; Zeiss or BZ-X710; Keyence, Japan), and were aligned using the microscope software (Zen software; Zeiss or BZ-X software; Keyence). Images were processed with ImageJ.
For Ki67+ OPC counting and GFP+ oligodendrocyte counting, two forebrain sections were analyzed for each mouse, and the results averaged. For OPC BrdU and fate tracing studies, two independent areas (one from each hemisphere) were analyzed from two brain sections for each mouse, for a total of four areas, and results were averaged. All cell countings were performed by an experimenter blinded to the animal’s genotype.
For Ki67+ OPC counting and GFP+ oligodendrocyte counting, two forebrain sections were analyzed for each mouse, and the results averaged. For OPC BrdU and fate tracing studies, two independent areas (one from each hemisphere) were analyzed from two brain sections for each mouse, for a total of four areas, and results were averaged. All cell countings were performed by an experimenter blinded to the animal’s genotype.
9
Quantifying γH2AX Signals in Replicating Cells
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To detect γH2AX signals, S-phase U2OS cells were pulsed-labeled with EdU and immuno-stained with anti-γH2AX antibody (Supplementary Table 2 ). After staining coverslips were mounted onto glass slides using VECTASHIELD Antifade Mounting Medium with DAPI (VectorLabs, H-1200) and imaged with a Keyence BZ-X800 microscope using the Keyence BZ-X software (Keyence). At least 300 EdU-positive cells were acquired.
10
Placental Calcification Quantification
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The von Kossa staining was performed as previously described in Speer et al. 2009 [31 (link)] with the inclusion of an optimally reduced 22 min von Kossa treatment. Placental tissue section from 8 normotensive pregnant women and 8 preeclamptic women were stained, and images of mounted sections were acquired on a Keyence BZ-X800 microscope using the Keyence BZ-X software (Keyence, Ozaka, Japan) at 4× magnification. File names were coded. Individual calcified lesions were imaged on a Nikon E800 Upright Microscope (Nikon Corp., Tokyo, Japan) at 2.5 and 10× magnification and file names were coded. Semi-quantitative analysis of calcified lesions was performed on all tissue sections using ImageJ/Fiji (NIH, Bethesda, MD, USA) histomorphometry software.
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