To determine the percentage of cells with aggregated (A), partially aggregated (PA) or dispersed (D) Met, GFP alone or EB3‐GFP transfected cells were stimulated with 0.5 nM HGF for 20 minutes and fixed with formaldehyde. The Met‐positive vesicles “at the MTs plus‐ends” have been scored at the vicinity of +end, stained with EB3‐GFP. The number of cells with aggregated, partially aggregated or dispersed Met were determined as previously described.33 Briefly, cells in each category have been counted under a scanning confocal microscope (LSM 510 Meta laser; Carl Zeiss, Inc.) with a ×100 plan Apo 1.4 NA objective and driven by ZEN LE software (Carl Zeiss, Inc.). In each experiment, a minimum of 20 fields was scored. See Figure S3D for representative images of each cell types.
Zen le software
Manufactured by Zeiss
Sourced in Germany
ZEN LE software is a comprehensive imaging and analysis platform developed by Zeiss. It provides a user-friendly interface for capturing, processing, and analyzing images from various Zeiss microscopes. The software offers essential tools for image acquisition, visualization, and basic image analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Axio observer, by Zeiss (3 mentions)
Lsm 510 meta laser, by Zeiss (2 mentions)
Rat anti cd31 antibody, by BD (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Rabbit anti s1p2r antibody, by Proteintech (2 mentions)
Lsm 710 confocal microscope system, by Zeiss (2 mentions)
Goat anti rat igg alexa 488, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa549, by Thermo Fisher Scientific (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Hb ap2100001, by Hanbio Biotechnology (1 mentions)
Normal goat serum (ngs), by Beyotime (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tunel apoptotic cell detection kit, by Roche (1 mentions)
15 protocols using zen le software
1
Quantifying Subcellular Met Localization
2
Immunohistochemical Analysis of S1P2 Receptor
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Mice were anesthetized with Avertin and were perfused with cold PBS and subsequently with 4% PFA in PBS solution. The brains were removed, post-fixed with 4% PFA for 24 h and transferred to 30% sucrose solution. Frozen brains were cut with a thickness of 30 μm in a cryostat. Slices were preserved in 30%PEG 30% sucrose PBS. The brain slices were washed three times with Tris-buffered saline (TBS) and were then blocked with TBS-blocking solution (1% bovine serum albumin, 0.2% skim milk and 0.3% Triton X-100 in TBS) for 1 h and incubated with the following primary antibodies in TBS-blocking solution overnight on a shaker at 4 °C. Rabbit anti-S1P2R antibody (1:100, Proteintech Group Inc., Chicago, IL) and rat anti-CD31 antibody (1:100, BD Biosciences, Pharmingen, San Jose, CA) were used as primary antibodies. Sections were washed three times with TBS and then they were incubated with donkey anti-rabbit IgG Alexa-594 and goat anti-rat IgG Alexa-488 (1:250, Life Technologies, Molecular Probes, Grand Island, NY). Brain slices were stained with DAPI for 7 min and were mounted onto slides. Samples were observed on a Zeiss LSM 510 Meta Confocal microscope (Carl Zeiss Inc.). Images were captured using Zen LE software (Carl Zeiss). The specificity of the S1PR2 antibody was confirmed by lack of signal in the S1pr2−/− brain sections.
3
Immunofluorescence Assay of Vav3 and VLRB in Lymphocytes
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Lymphocyte cells were isolated from animals before and after treated with LPS or PHA, and were suspended in 1.5 mL Eppendorf tubes and fixed with paraformaldehyde solution (4% in phosphate buffer saline (PBS)) for 20 min at room temperature. Then, Immunofluorescence assay was performed according to the description of Han et al. [38 (link)] with a rabbit anti-recombinant lamprey Vav3 poly-clonal antibody (1000-fold) and a mouse anti-VLRB mono-clonal antibody (1000-fold) [46 (link)]. Cells were observed by a Zeiss LSM710 Confocal Laser Scanning Microscope (Oberkochen, Germany) and each type of cells (including big and round VLRB+ plasmacytes and small VLRB+ lymphocytes) were counted and analyzed in 5 microscope fields by using Zeiss ZEN LE software.
4
Endostar Induces Autophagy in Cells
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Cells seeded at a density of 3×105 cells/well into six-wells plates were treated with a specific dose (25, 50, 100 and 200 µg/ml) of Endostar for 24 h at 37°C, then incubated with LysoTracker (Invitrogen; Thermo Fisher Scientific, Inc.,) for 90 min at 37°C. Subsequently, cells were washed twice with PBS, fixed with 4% paraformaldehyde at 37°C for 2 h and permeabilized with 1% CHAPS buffer (150 mM NaCl, 10 mM HEPES, 1.0% CHAPS) at room temperature for 15 min. Then, cells were incubated with an anti-LC3 antibody (dilution, 1:1,000; cat. no. 4455) for 2 h at 37°C, washed with PBS three times and incubated with FITC-conjugated goat anti-rabbit IgG (dilution, 1:200; cat. no. A0562) for 1 h at 37°C. Next, the cell nuclei were stained using DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min at 37°C. Samples were examined under a Zeiss LSM 710 fluorescence microscope system (Carl Zeiss AG, Oberkochen, Germany; magnification, ×1,000). Images were processed using ZEN LE software (version 2.0; Carl Zeiss AG).
For the quantification of LC3-positive cells, 150–200 cells were randomly selected from the acquired image and counted. Cells with >5 dots of specific red signals were considered to be autophagic (LC3-positive).
For the quantification of LC3-positive cells, 150–200 cells were randomly selected from the acquired image and counted. Cells with >5 dots of specific red signals were considered to be autophagic (LC3-positive).
5
Monitoring Autophagy Flux with Tandem Fluorescent LC3
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A tandem monomeric RFP-GFP-tagged LC3 (tfLC3) was used to monitor autophagy flux as previous reported. For evaluating tandem fluorescent LC3 puncta, 48 h after tfLC3 transfected alone, or with siRNAs/plasmid cotransfected, cells were washed with 1 × PBS, and then sent out for confocal microscope analysis directly. Samples were examined under a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Oberkochen, Germany) and images were processed with ZEN LE software (Carl Zeiss, Oberkochen, Germany)
6
Immunofluorescence Staining of Cultured Cells
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Cells were treated with or without drugs as indicated, fixed with cold methanol for five minutes at −20 °C. Cells were then washed three times with PBS and blocked with blocking buffer (2.5% BSA + 0.1% Triton X-100 in PBS) at room temperature for 1 h. Cells were incubated with primary antibodies at 4 °C overnight, washed five times with PBS buffer and then incubated with appropriate secondary antibodies conjugated to Alexa488 and Alexa549 (Molecular Probes) for 2 h at room temperature. DNA was stained with DAPI. Slides were examined under a Zeiss AX10 microscope system (Carl Zeiss, Germany) and images were processed with ZEN LE software (Carl Zeiss).
7
Quantifying Focal Adhesion Dynamics
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Stable adhesions were selected from the leading edge of migrating cells. Images of Paxillin-expressing cells (n = 20) were acquired every 4 s for 2 min. Background-corrected fluorescence intensity images were used to measure small adhesion sites and FA size and number (Zaoui et al., 2008 (link)). FRAP experiments were performed on a confocal microscope (LSM710; Carl Zeiss) with a 100× plan Apo 1.4 NA objective. For FAs, EGFP fluorescence (from 10 cells and 30 adhesions for stimulated cells; 20 cells and 40 adhesions for unstimulated cells) was eliminated by 30 bleach cycles at 100% intensity of the 488-nm argon laser. Recovery curves generated from photobleached areas of the same size and locations were sampled every 1 s for 2 min and corrected for overall photobleaching, and then used to calculate percentage of fluorescence recovery by Zen LE software (Carl Zeiss).
8
Immunofluorescence Staining of Mouse Brain
9
Immunofluorescence Staining of Microtubules
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Cells grown on collagen‐coated coverslips were fixed in 4% formaldehyde at room temperature or 100% methanol at −20°C for MTs staining and permeabilized in 0.2% Triton X‐100 before the addition of antibodies. Antibodies used for IF were against the following: Met (AF276 from R&D Systems); α‐tubulin (Boehringer, Ingelheim), CLIP‐170, GFP mouse (Roche, Inc.). Secondary antibodies and Alexa Fluor 488, 546 and 633, and phalloidin were obtained from Molecular Probes. Images were recorded with a scanning confocal microscope (LSM 510 Meta laser; Carl Zeiss, Inc.) with a ×100 plan Apo 1.4 NA objective and driven by ZEN LE software (Carl Zeiss, Inc.).
10
CALI of RhoA and Ran Dynamics
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For chromophore-assisted laser or light inactivation (CALI) experiments, TOV-112D cells co-expressing GFP-RhoA and Ran-KillerRed or GFP-RhoA-CCKVL and KillerRed empty vector were irradiated for 1 min with green light (×100 plan Apo 1.4 NA objective, 515–560 -nm transmitted light at 18 W/cm2) to bleach KillerRed fluorescence. After bleaching, green fluorescence was recorded every second over a period of 5 min with a scanning confocal microscope (ZEISS Axio Observer; Carl Zeiss, Inc.) with a ×100 plan Apo 1.4 NA objective and driven by ZEN LE software (Carl Zeiss, Inc.).
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