The immunofluorescence analysis was performed in the following way. Round colonies of EOS-selected, YAP-treated cells were placed on 30-mm glass bottom dishes (MATSUNAMI, Tokyo, Japan, D11530H) and grown for 24 hours in YAP medium. On the day of immunostaining, the colonies were washed with PBS, fixed with 4% PFA (Wako, Tokyo, Japan,163-20145) for 15 min at room temperature, and permeabilized with 0.5% Triton (Sigma-Aldrich, X100-5ML, Merck, Darmstadt, Germany) in PBS with 10% FBS addition for 30 min. The following antibodies were applied: primary anti-SUSD2 (Sigma, HPA004117-100UL, Merck, Darmstadt, Germany) and anti-KLF17 (Sigma, HPA024629-100UL, Merck, Darmstadt, Germany), in PBS with 10% FBS addition, for 1 hour at room temperature. After 1 hour, the cells were washed with PBS twice and incubated with secondary antibodies: goat anti-rabbit IgG H&L (Alexa Fluor® 594) (Abcam, Tokyo, Japan, ab150080, 1/500 dilution) in PBS with 10% FBS addition, for 1 hour at room temperature. Then, the cells were washed 4 times with PBS, and 2 mL of PBS per dish was added for imaging. Imaging was performed using a KEYENCE BZ-9000 BIOREVO fluorescent microscope.
Biorevo bz 9000 fluorescent microscope
Manufactured by Keyence
Sourced in Japan
The BIOREVO BZ-9000 is a fluorescent microscope designed for advanced biological research. It features high-resolution imaging capabilities, supporting a range of fluorescent labeling techniques. The microscope is equipped with LED illumination and a motorized stage for efficient sample handling. Its core function is to provide detailed visualization and analysis of fluorescently-labeled biological specimens.
Automatically generated - may contain errors
Lab products found in correlation
30 mm glass bottom dishes, by Matsunami (2 mentions)
X100 5ml, by Merck Group (2 mentions)
Hpa004117 100ul, by Merck Group (2 mentions)
Goat anti rabbit igg h l alexa fluor 594, by Abcam (2 mentions)
Anti pax6 polyclonal antibodies, by BioLegend (2 mentions)
Falcon cultureslides, by Corning (1 mentions)
Anti human e cadherin antibody, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Nacalai Tesque (1 mentions)
Alexa 555 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Slowfade, by Thermo Fisher Scientific (1 mentions)
8 protocols using biorevo bz 9000 fluorescent microscope
1
Immunofluorescence Analysis of YAP-Treated Cells
2
Immunocytochemistry of Epithelial and Mesenchymal Markers
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The cells were cultivated for 72 h, with subsequent fixation with 4% paraformaldehyde in chamber slides (Falcon® Culture Slides; Corning, NY, USA and Sarstedt, Nümbrecht, Germany). The cells were permeabilized with 0.1% Triton-X and sequentially treated with goat serum, primary monoclonal mouse anti-human CK 7 antibody (DAKO Corporation, Clone OV-TL 12/30, 1:200 dilution), primary monoclonal rabbit anti-human E-cadherin antibody (Cell Signaling Technology, Frankfurt/Main, Germany, #3195, 1:200 dilution), primary monoclonal mouse anti-human vimentin antibody (Abcam, Cambridge, UK, V1-RE/1, ab3974,1:200 dilution), anti-Mouse Alexa Fluor® 594 secondary antibody (Abcam, #150116, 1:500 dilution), and anti-Rabbit Alexa Fluor® 488 secondary antibody (Abcam, #150077, 1:500 dilution). Appropriate negative controls were performed. Coverslips were mounted with Fluoromount-G medium including DAPI (Southern Biotech, Huntsville, AL, USA). The cells were visualized using a BZ-9000 (BIOREVO) fluorescent microscope (Keyence, Osaka, Japan) with a 20× PlanFluor EL NA 0.45 Ph1 objective lens under 0.285 s of exposure time for CK7 and 0.2 s of exposure time for E-cadherin and vimentin.
3
Immunofluorescence Analysis of YAP-treated Cells
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The immunofluorescence analysis was performed in the following way. Round colonies of EOS-selected, YAP-treated cells were placed on 30-mm glass bottom dishes (MATSUNAMI, Tokyo, Japan, D11530H) and grown for 24 hr in YAP medium. On the day of immunostaining, the colonies were washed with PBS, fixed with 4% PFA (Wako, 163-20145) for 15 min at room temperature, and permeabilized with 0.5% Triton (Sigma-Aldrich, X100-5ML, Merck) in PBS with 10% FBS addition for 30 min. The following antibodies were applied: primary anti-SUSD2 (Sigma, HPA004117-100UL, Merck) and anti-KLF17 (Sigma, HPA024629-100UL, Merck), in PBS with 10% FBS addition, for 1 hr at room temperature. After 1 hr, the cells were washed with PBS twice and incubated with secondary antibodies: goat anti-rabbit IgG H&L (Alexa Fluor® 594) (Abcam, Tokyo, Japan, ab150080, 1/500 dilution) in PBS with 10% FBS addition, for 1 hr at room temperature. Then, the cells were washed 4 times with PBS, and 2 mL of PBS per dish was added for imaging. Imaging was performed using a KEYENCE BZ-9000 BIOREVO fluorescent microscope.
4
PAX6 Immunofluorescence Staining Protocol
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Cell staining was performed as previously described51 (link). Briefly, cells were cultured on glass coverslips, fixed in 4% paraformaldehyde in phosphate-buffered (PBS, pH 7.4) for 15 min at room temperature, and then incubated with anti- PAX6 polyclonal antibodies (1:100, Biolegend, San Diego, CA, USA) overnight at 4 °C. Then, the cells were incubated with Alexa 555-conjugated secondary antibodies (1:200, Thermo Fisher Scientific) for 1 h at room temperature. Nuclei were counterstained with DAPI (Nacalai Tesque). Fluorescence images were obtained using a BIOREVO BZ-9000 fluorescent microscope (Keyence, Osaka, Japan). PAX6 positive nuclei were counted using the ImageJ software (NIH, Bethesda, MD, USA).
5
Subcellular Localization of OsCCD4b-GFP
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For the construction of OsCCD4b-GFP, the ORF of OsCCD4b (Os12g24800) without the stop codon was amplified by PCR and subcloned into the corresponding site on the pE7133-GFP vector [55 (link)], fusing GFP in-frame to the C-terminus of OsCCD4b. The OsCCD4b-GFP fusion protein was introduced into rice protoplast cells using polyethylene glycol 3350, according to Bart et al. [56 (link)]. Localization analysis of OsCCD4b was performed as previously described by Kiryu et al. [23 (link)]. GFP fluorescence was observed using a BIOREVO BZ-9000 fluorescent microscope (Keyence, Osaka, Japan) equipped with a GFP-specific filter unit.
6
Immunocytochemistry for Pax6 Expression
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Cell staining was performed as previously described (Hirata et al., 2014) . Cells, cultured on glass coverslips, were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature. The fixed cells were incubated with anti-PAX6 polyclonal antibodies (1:100; Biolegend, San Diego, CA, USA) overnight at 4°C. Finally, they were incubated with Alexa594-conjugated secondary antibodies (1:200; Life Technologies) for 1 hr at room temperature. The cells were enclosed in SlowFade (Life Technologies) and examined using a BIOREVO BZ-9000 fluorescent microscope (Keyence, Osaka, Japan).
7
Myotube Fusion Index Quantification
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Nuclei of C2C12 myotubes were fixed and stained using propidium iodide. Each well was photographed in six randomly selected regions at × 20 magnifications at day 3 or day 5 post-differentiation induction using a BIOREVO BZ-9000 fluorescent microscope (Keyence, Osaka, Japan). The number of nuclei incorporated in myotubes and the total number of nuclei were scored. Fusion index was calculated as the percentage of total nuclei incorporated in myotubes. Diameter of myotubes was measured using BIOREVO BZ-9000 software (Keyence).
8
Coronal Spinal Cord Immunostaining Protocol
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Spinal cord coronal sections were dried at room temperature for 10–15 min, washed with Phosphate Buffer Saline (PBS) for 5 min, and post-fixed with 4% PFA for 10 min. The sections were blocked with 20% Bovine Serum Albumin (BSA) for 2 h at room temperature, and incubated at 4 °C in antibody solutions containing either mouse anti-Neuronal Nuclei (NeuN) monoclonal antibody (clone A60, 1:100; from Millipore, Billerica, MA, USA), mouse anti-adenomatous polyposis coli (APC)-7 monoclonal antibody (clone CC-1, 1:200; from Calbiochem, San Diego, CA, USA), and rabbit polyclonal anti-5HT antibody (1:500; from Abcam, Cambridge, MA, USA) to examine the immunoreactivity (IR) and cell numbers of neurons, mature oligodendrocytes, and IR of serotonin. On the next day, the sections were incubated with Alexa Fluor®488 or 594-conjugated donkey anti-mouse (1:500; from Invitrogen, Carlsbad, CA, USA). Control slides were incubated without primary antibodies to further confirm the specificity of the IR. The slides were examined with a BIOREVO BZ-9000 fluorescent microscope (Keyence, Itasca, IL, USA)
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