293T cells were seeded on glass‐bottom cell culture dishes (NEST) and IDR‐mCherry‐CRY2 or R>A mutant plasmids were transfected into the cells. One day after transfection, images were acquired on ZEISS LSM900 confocal microscope with a 63×, 1.4 NA oil immersion objective lens using ZEN 3.0 software and a CCD camera. To activate the self‐association of CRY2, cells were excited with a 488 nm laser for 6 s every 6 s, during which the mCherry signal was recorded. To analyze the co‐condensation between CRTC2‐IDR and P‐TEFb, IDR‐mCherry‐CRY2 plasmids were transfected into 293T cells. One day after transfection, cells were excited with a 488 nm laser for 5 min. Then incubated with primary antibody against CDK9 (Santa Cruz, sc‐13130) or CycT1 (Santa Cruz, sc‐8127). The samples were imaged by ZEISS LSM900 confocal microscope with a 63×, 1.4 NA oil immersion objective lens using LAS V4.4 software and a CCD camera.
Lsm 900
Manufactured by Zeiss
Sourced in Germany, United States, Italy, Japan, Canada
The LSM 900 is a confocal laser scanning microscope designed for high-resolution imaging. It utilizes laser excitation and a pinhole system to achieve optical sectioning, allowing for the visualization of three-dimensional structures within samples.
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Lab products found in correlation
Airyscan 2, by Zeiss (36 mentions)
Lsm 880, by Zeiss (24 mentions)
Lsm 700, by Zeiss (17 mentions)
Lsm 710, by Zeiss (14 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (13 mentions)
Zen blue software, by Zeiss (10 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (9 mentions)
Lsm 800, by Zeiss (8 mentions)
Zen software, by Zeiss (7 mentions)
Efficient navigation zen , by Zeiss (7 mentions)
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Lsm 780, by Zeiss (6 mentions)
Zen 3, by Zeiss (6 mentions)
Airyscan, by Zeiss (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Fluo 4 am, by Thermo Fisher Scientific (5 mentions)
Hepes, by Merck Group (4 mentions)
Mitotracker green, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (4 mentions)
Plan apochromat 63 1.4 oil dic m27, by Zeiss (3 mentions)
490 protocols using lsm 900
1
Visualizing CRY2-Mediated Condensation
2
Zebrafish and Mouse Imaging Protocol
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Zebrafish images were acquired on a Zeiss LSM 900 with AiryScan 2 using a 40× water-immersion LD C-Apochromat [numerical aperture (NA) 1.1] or 63× oil-immersion Plan Apochromat (NA 1.4) with laser lines 405, 488, 561, 640 nm. Embedding was performed using glass-bottom dishes and 1% low-melting-point agarose (Sigma-Aldrich, A9414), which was covered following solidification with E3.
Mouse data were acquired on a Zeiss LSM900 with AiryScan 2 using a Plan-Apochromat 63×/1.40 Oil DIC f/ELYRA.
Sampling frequency was assessed for Tg(TP1bglob:VenusPest)s940 using the Nyquist sampling rules (https://svi.nl/NyquistCalculator ; Eqns 1-3): For calculations, an Array Detector was used as microscope type, with the following parameters/settings: numerical aperture, 1.3; excitation wavelength, 488; emission wavelength, 520; number of excitation photons, 1; lens immersion refractive index, 1.338. Zeiss AiryScan was used as Array Detector model. This resulted in a Nyquist sampling of: x, 46 nm; y, 46 nm; z, 119 nm.
Mouse data were acquired on a Zeiss LSM900 with AiryScan 2 using a Plan-Apochromat 63×/1.40 Oil DIC f/ELYRA.
Sampling frequency was assessed for Tg(TP1bglob:VenusPest)s940 using the Nyquist sampling rules (
3
Visualizing NF-κB and Autophagy in HUVECs
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Immunofluorescence staining assay was used for the detection of NF-κB p65 nuclear translocation and LC3B transformation. HUVECs were transfected with pcDNA 3.1-MALAT1 and stimulated with TNF-α (10 ng/mL) in the presence or absence of bafilomycin for 24 h. HUVECs were fixed with 4% paraformaldehyde for 1 h and permeabilised with 0.5% Triton X-100 for 30 min, and subsequently blocked with bovine serum albumin for 1 h. HUVECs were incubated with anti-NF-κB p65 antibody (Beyotime) at 4 °C overnight. After gentle washing, anti-rabbit Cy3 Ab was added to the fixed and permeabilised cell monolayers and incubated at 37 °C for 1 h. The reaction was stopped using 4’,6-diamidino-2-phenylindole. The cells were then visualised and images were captured using a confocal microscope (LSM 900, Zeiss, Germany).
To detect autophagy, immunofluorescence staining of LC3B-positive autophagosomes was carried out using an LC3B antibody kit for autophagy (Cell Signalling Technology). The staining was performed according to the manufacturer's instructions. The number of endogenous LC3B puncta was evaluated by confocal microscopy (LSM 900; Zeiss, Germany).
To detect autophagy, immunofluorescence staining of LC3B-positive autophagosomes was carried out using an LC3B antibody kit for autophagy (Cell Signalling Technology). The staining was performed according to the manufacturer's instructions. The number of endogenous LC3B puncta was evaluated by confocal microscopy (LSM 900; Zeiss, Germany).
4
Multimodal Imaging of Cellular Dynamics
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Fixed samples were imaged with a 34-channel Zeiss LSM 710 or LSM 900 confocal microscope equipped with an Airyscan detector and with a Plan Apo 63× numerical aperture (NA) 1.4 oil immersion objective. Wide-field images were acquired on a Zeiss LSM 900 microscope equipped with an sCMOS Prime 95B camera and a Plan Apo 40× NA 1.3 oil immersion objective.
2D STED super-resolution imaging was performed with a STEDYCON (Abberior Instruments GmbH) mounted on an upright microscope base (Zeiss AxioImager Z.2) equipped with a Zeiss Plan Apochromat 100×, NA 1.4 oil immersion objective actuated by a z-piezo with 100-μm range (Physik Instruments). In STED mode, 100% of a maximum nominal STED laser output of 1.25 W (775 nm) was used, leading to a theoretical spatial resolution of 35 to 40 nm. STAR RED and STAR ORANGE were excited by pulsed laser sources at 640 and 561 nm, respectively. Pinhole size was 1.13 Airy units at 650 nm. Gated detection in single-photon counting mode was performed using Avalanche photodetectors.
For live-cell imaging, the previously described Zeiss LSM microscopes were equipped with stage-top incubation chambers, allowing temperature and CO2 control. Cells were imaged 30 min after seeding on nanostructured substrates. Several time series of 30 min were then recorded over the course of 2 hours. Observations were made at 37°C and 5% CO2.
2D STED super-resolution imaging was performed with a STEDYCON (Abberior Instruments GmbH) mounted on an upright microscope base (Zeiss AxioImager Z.2) equipped with a Zeiss Plan Apochromat 100×, NA 1.4 oil immersion objective actuated by a z-piezo with 100-μm range (Physik Instruments). In STED mode, 100% of a maximum nominal STED laser output of 1.25 W (775 nm) was used, leading to a theoretical spatial resolution of 35 to 40 nm. STAR RED and STAR ORANGE were excited by pulsed laser sources at 640 and 561 nm, respectively. Pinhole size was 1.13 Airy units at 650 nm. Gated detection in single-photon counting mode was performed using Avalanche photodetectors.
For live-cell imaging, the previously described Zeiss LSM microscopes were equipped with stage-top incubation chambers, allowing temperature and CO2 control. Cells were imaged 30 min after seeding on nanostructured substrates. Several time series of 30 min were then recorded over the course of 2 hours. Observations were made at 37°C and 5% CO2.
5
Characterizing Oxidative Stress and Mitochondrial Superoxide
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Oxidative stress changes are represented by reactive oxygen species (ROS). The fluorescence intensity of DCFH-DA is the most commonly used method to detect intracellular ROS levels. HBE cells were seeded in confocal dishes overnight. Fe3O4/GO (0, 100, 200 μg/ml) in DMEM was added and then coincubated after 24 h. Then, it was cleaned with PBS three times, then prepared DCFH-DA staining solution was added and incubated for 15 min. It was cleaned with PBS three times, observed, and analyzed by using a confocal microscope (LSM 900, ZEISS, Germany) (Ex: 505 nm Em: 525 nm).
Mitosox red is a mitochondrial superoxide indicator. HBE cells were seeded in confocal dishes overnight. Fe3O4/GO (0, 100, 200 μg/ml) in DMEM was added and then coincubated after 24 h. Then, it was cleaned with PBS three times, prepared Mitosox red and DAPI staining solution was added and incubated for 15 min. It was cleaned with PBS three times, observed, and analyzed via a confocal microscope (LSM 900, ZEISS, Germany) (Mitosox red, Ex: 510 nm Em: 580 nm; DAPI, Ex: 350 nm Em: 461 nm).
Mitosox red is a mitochondrial superoxide indicator. HBE cells were seeded in confocal dishes overnight. Fe3O4/GO (0, 100, 200 μg/ml) in DMEM was added and then coincubated after 24 h. Then, it was cleaned with PBS three times, prepared Mitosox red and DAPI staining solution was added and incubated for 15 min. It was cleaned with PBS three times, observed, and analyzed via a confocal microscope (LSM 900, ZEISS, Germany) (Mitosox red, Ex: 510 nm Em: 580 nm; DAPI, Ex: 350 nm Em: 461 nm).
6
Nephrocyte Function Screening Assay
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The ANF-RFP uptake assay was used to screen nephrocyte function in the Mhc-ANF-RFP, Hand-GFP, Dot-Gal4, UAS-APOL1-G1; UAS-RNAi and Mhc-ANF-RFP, Hand-GFP, Dot-Gal4, UAS-APOL1-G0; UAS-RNAi transgenic lines. Adult fly nephrocytes were dissected and kept in artificial hemolymph (fly blood), and then fixed in 4% paraformaldehyde in 1X phosphate buffered saline (1XPBS) for 10 min. ANF-RFP uptake by nephrocytes was assayed by fluorescence confocal microscopy (ZEISS LSM 900; see details below). Nephrocyte number and cell size were determined by fluorescent confocal microscopy (ZEISS LSM 900; see details below), then compared to wildtype and APOL1-G1 transgenic flies without RNAi.
7
Live Fluorescence Imaging of Medaka Hatchlings
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Live fluorescence imaging was performed using a stereomicroscope (Nikon SMZ18) equipped with the NIS-Elements BR 3.0 software or confocal microscopes (Zeiss Meta 500; Olympus FluoView FV3000; Zeiss LSM900). Medaka hatchlings (8 to 23 dpf) were anaesthetized with 0.005% ethyl 3-aminobenzoate methane sulfonate (Tricaine; Sigma MS-222) and mounted in 1.5% low-melting-point agarose on a glass bottom petri dish. Confocal pictures were taken using 405, 488, 543 or 633 nm laser lines for CFP, GFP, mCherry and Cy5 fluorescent signals, respectively. Time-lapse imaging was performed with Olympus FV3000 or Zeiss LSM900 microscopes by imaging the region of interest for 15-20 hours with 5-10 mins intervals. Imaging data were processed using Olympus FV31S-SW 2.1.1.98, Bitplane Imaris 9.0, ImageJ and Adobe Photoshop CC 2018 software.
8
Quantifying FATP1 and CD37 Colocalization
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Antibody stainings for flow cytometry (MACS Quant, Miltenyi Biotec, Bergisch Gladbach, Germany) and confocal microscopy (LSM900, Zeiss, Jena, Germany) were performed on cells fixed with 2% paraformaldehyde and blocked with 5 µM FcBlock (Bd Biosciences, San Jose, CA, USA). Antibodies directed against FATP1 (308420, conjugated) (R&D Systems, Minneapolis, MN, USA, IC3304R), CD36 (877302, conjugated) (Novus Biologicals, Centennial, CO, USA, MAB19553AF647) and CD37 (HH1, unconjugated) (Santa Cruz Biotechnology, Dallas, TX, USA, sc-18881) were added as 10 µg/mL in PBS and stained for 30 min at RT in the dark. Isotype controls were added in similar concentrations. A secondary antibody against CD37 was used as 2,5 µg/mL in PBS and stained for 30 min at RT in the dark. For colocalization studies (LSM900, Zeiss, Jena, Germany), the Coloc2 function in ImageJ was used (Version 1.53 g)93 (link). Proximity ligation assays (PLA) were performed with FATP1 and CD37 antibodies according to the manufacturer’s instructions (Merck, Darmstadt, Germany). In addition, a second FATP1 antibody (polyclonal) (Novus Biologicals, Centennial, CO, USA, NBP2-69016) was used at 10 µg/mL as a positive control. CD37KO BJAB cells (generated by CRISPR/Cas9 knockout, described in92 (link)) were used as a negative control in these experiments. Analysis was performed with ImageJ42 (link),94 (link).
9
Immunostaining and Live Imaging of Oocytes
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Oocytes without cumulus cells were fixed at room temperature (RT) in 4% paraformaldehyde/PBS for 30 min, permeabilized in 1% Triton X‐100/PBS for 8 h, and blocked in 1% BSA/PBS for 1 h. Then denuded oocytes were incubated with γH2AX antibody (Cell Signaling Technology, Danvers, MA; 1:100), LCA‐FITC (Sigma‐Aldrich; 1:100), α‐tubulin‐FITC antibody (Sigma‐Aldrich; 1:200), phalloidin‐TRITC (Sigma‐Aldrich; 1:100) or anti‐human ovastacin antibody (Jurrien Dean lab, NIH; 1:100) at 4°C overnight. Oocytes were next washed in PBST, followed by incubation with the secondary antibodies for 1 h and 10 μg/ml propidium iodide (PI) for 10 min at RT. Lastly, oocytes were mounted on the glass slides for imaging using laser confocal microscope (LSM 900, Carl Zeiss, Germany).
For live staining, oocytes were incubated with MitoTracker Red CMXRos (ThermoFisher Scientific; 1:2000), MitoProbe JC‐1 (ThermoFisher Scientific; 1:100), Dichlorofluorescein Diacetate (Beyotime, Huangzhou, China; 1:800) or Annexin‐V‐FITC (Beyotime, Huangzhou, China; 1:10) at 38.5°C for 30 min, followed by imaging using laser confocal microscope (LSM 900, Carl Zeiss, Germany).
For live staining, oocytes were incubated with MitoTracker Red CMXRos (ThermoFisher Scientific; 1:2000), MitoProbe JC‐1 (ThermoFisher Scientific; 1:100), Dichlorofluorescein Diacetate (Beyotime, Huangzhou, China; 1:800) or Annexin‐V‐FITC (Beyotime, Huangzhou, China; 1:10) at 38.5°C for 30 min, followed by imaging using laser confocal microscope (LSM 900, Carl Zeiss, Germany).
10
Immunostaining Protocols for Drosophila Ovaries
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For immunostaining, ovaries were dissected and fixed in 5.3% PFA in PBS for 10 min and washed three times for 20 min in 0.2% TritonX-100 in PBS (PBX). After incubating with blocking buffer (4% BSA, 0.2% PBX) for 40 min, ovaries were incubated with primary antibodies (Supplementary Table 2 ). overnight at 4 °C followed by three washes in PBT for 15 min each. Incubation with Alexa Fluor-secondary antibodies diluted in 0.4% BSA in PBS was carried out overnight at 4 °C followed by three washes in PBX for 15 min each. Nuclei were stained with DAPI (1:500 in wash buffer). Samples were mounted in mounting media (Fluoro-KEEPER, Nacalai). The images were obtained with a Zeiss LSM900 or LSM780 under ×63 or ×40 magnification. GSC counting was performed with an Olympus Axiovert. Cap cell counting was performed with a Zeiss LSM900. A chi-square test was used to compare pH3 labeled GSCs (****P ≤ 0.001, ***P ≤ 0.005, **P ≤ 0.01, n.s., nonsignificant (P > 0.05)).
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