The resulting stained specimens were examined and photo-documented using the Keyence Biorevo BZ-9000 microscope (Keyence, Neu-Isenburg, Germany) and its corresponding BZII-Viewer-Analyzer program (Brightfield HF and Phako, microscope position 2 Plan Apo Na.10; Keyence, Neu-Isenburg, Germany) as previously described (Figure 3 ) [22 (link)]. The pictures were analyzed using the application Hybrid-Cell-Count and Brightfield & Single extraction. With the subsequent adjustment of tolerance and transparency, the ratio of strongly stained sections of histological tissue to less stained areas could be calculated as a portion (in percentage) of the whole sample.
Biorevo bz 9000 microscope
Manufactured by Keyence
Sourced in Japan, Germany, United States
The BIOREVO BZ-9000 is a high-performance microscope designed for laboratory use. It features a range of advanced optics and imaging capabilities to facilitate detailed observation and analysis of samples. The core function of the BIOREVO BZ-9000 is to provide clear, high-resolution images of specimens under study.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
Bz 2 analyzer software, by Keyence (6 mentions)
Hasmc, by Lonza (4 mentions)
β glycerophosphate, by Merck Group (3 mentions)
Dynamic cell count bz h1c software, by Keyence (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Ascorbic acid phosphate, by Merck Group (2 mentions)
Dexamethasone (dex), by AppliChem (2 mentions)
Ultracut e, by Reichert Technologies (2 mentions)
Nonspecific staining blocking reagent, by Agilent Technologies (2 mentions)
Histofine simple stain kit, by Nichirei Biosciences (2 mentions)
Imsol mount, by Immunologic (2 mentions)
Ab36861, by Abcam (2 mentions)
Bz 2 analyzer, by Keyence (2 mentions)
Hasmc, by Merck Group (2 mentions)
Ang 2, by Merck Group (2 mentions)
Millicell ez slide, by Merck Group (2 mentions)
Smgm 2, by Lonza (2 mentions)
Paraformaldehyde solution, by Fujifilm (2 mentions)
Mmp13, by Abcam (2 mentions)
116 protocols using biorevo bz 9000 microscope
1
Quantitative Histological Analysis
2
Immunohistochemical Quantification of ALDH2 and γ-H2AX in Esophageal Epithelium
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Immunohistochemistry was performed as described43 (link). Primary antibodies and the titers used were as follows: rabbit polyclonal anti-ALDH2 (Ag7452; 15310-1-AP; Proteintech Group, Inc., Chicago, IL, USA; 1:500); rabbit monoclonal anti-phospho-histone H2AX (Ser139; 20E3; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:480). Immunostained tissues were assessed using a Keyence BIOREVO BZ-9000 microscope (Keyence Corp., Osaka, Japan). For the staining of ALDH2, we defined the sample as positive when more than 50% of the cells were stained with anti-ALDH2 in the basal and parabasal layer of the oesophageal epithelium together. We present the ratio of cells positive for phosphorylated histone H2AX (γ-H2AX) to the total number of cells, determined using a Hybrid Cell Count BZ-H2C system (Keyence Corp.), as a staining index.
3
Histochemical GUS Staining of Leaves
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Barley and N. benthamiana leaves were harvested for GUS staining three and 2 days, respectively, after bombardment/or Agrobacterium infiltration. The leaves were submerged in 5-bromo-4-chloro-3-indolyl-ß-d -glucoronide cyclohexylammonium (X-Gluc) staining solution (42.3 mM NaH2PO4, 57.7 mM Na2HPO4, 10 mM EDTA, 20 % methanol, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 × 3 H2O, 1 mg/mL X-Gluc, 0.1 % Triton X-100) and vacuum was applied three times for 10 min. Then, the material was incubated at 37 °C overnight. Afterwards, the leaves were bleached in 80 % EtOH at room temperature for at least 2 days. The leaves were screened for blue-stained cells by bright field microscopy; photographs were taken with the Keyence Biorevo BZ9000 microscope (Keyence Corporation, Neu-Isenburg, Germany).
4
Liver Histopathology Evaluation Protocol
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Liver histopathology was performed according to a previously described protocol [34 (link)]. In brief, histopathological specimens were prepared by fixing livers isolated from mice with 10% formalin neutral buffer solution followed by paraffin embedding using common methods. We cut 3-μm thick microtome sections and hematoxylin-eosin (H&E) staining was performed to observe necrotic cell death in APAP overdose livers. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of liver samples was performed to observe hepatocyte death using an ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore, Billerica, MA, USA) according to the protocol supplied by the manufacturer. Images of stained sections were captured by a KEYENCE BIOREVO BZ-9000 microscope (Keyence Corporation, Osaka, Japan).
5
Tumor Immunohistochemical Staining and Quantification
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Following excision, tumors were snap frozen in liquid nitrogen and subsequently stored at − 80°C. Sections were used for staining towards CD31, CD34, Ki67 and CAIX using standard procedures [22] . The following antibodies, diluted in a commercial antibody diluent (DAKO, Hamburg, Germany), were used: anti-Ki-67 antigen (clone MIB-1, DAKO), final dilution 1:25; anti-CD31 (clone SZ31, DAKO), final dilution 1:100; anti-CD34 (clone MEC14.7, Linaris, Dossenheim, Germany), final dilution 1:200 and anti-carbonic anhydrase 9 (CA9, CAIX, clone GT12, GeneTex, Irvine, USA), final dilution 1:500. After incubation with the primary antibody for 1 hour at room temperature, the slides were washed in PBS and incubated with the following secondary antibodies: goat anti-mouse-HRP (DAKO), final dilution 1:100 or rabbit anti-rat-HRP (DAKO), final dilution 1:100. After development in 5% 3,3′-diaminobenzidine (DAKO) and counterstaining with haematoxilin, the sections were dehydrated in graded ethanol and embedded in Vitro Clud (Langenbrinck, Emmendingen, Germany). Stained slides were photographed at 20x magnification with a Keyence Biorevo BZ-9000 Microscope (Keyence Corporation, Osaka, Japan) applying Z-stack technology to improve the quality of images. The number of stained cells per section was quantified by using measurement module BZ-H3C (Hybrid Cell Count Vers.1.1, Keyence).
6
DAF-16 Localization in C. elegans
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The C. elegans strain TJ356, having daf-16 fused to GFP, was utilized for observing the localization of DAF-16 transcription factor. L1 synchronized larvae were divided into seven groups (30–50 worms each) and treated as described above using S-medium + E. coli OP50 at 20 °C. TJ356 worms were investigated after 24 h using fluorescence microscopy. Worms were fixed on glass slides using 10 mM sodium azide for paralysis. A BIOREVO BZ-9000 microscope, equipped with a mercury lamp, at an excitation and emission wavelengths of λex 480/20 nm and λem 510/38 nm (Keyence Deutschland GmbH, Neu-Isenburg, Germany) and an Axiostar Plus 37081 fluorescence microscope (Carl Zeiss) were used to view the nematodes. Pictures of 30–40 worms were randomly viewed for analysis using 20X objective lens and a constant time of exposure. Worms were counted after dividing them according to the localization of DAF-16::GFP to cytosolic, nuclear, or intermediate localization. Analysis and comparing nuclear results percentage between different groups were done as described above. Three independent sets of the experiment were performed and averaged.
7
Bioprint and Jawbone Model Cultivation
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For material testing, bioprints were cultivated in growth medium for 28 days in 24-well ultra-low attachment multiple well plates, while medium was exchanged three times a week.
For jawbone models, constructs were cultivated in growth medium or mineralisation medium [growth medium supplemented with 10 mM β-glycerophosphate (Sigma-Aldrich, Saint Louis, USA), 10 nM dexamethasone (AppliChem, Darmstadt, Germany) and 284 µM ascorbic acid phosphate (Sigma-Aldrich, Saint Louis, USA)] for up to 28 days in 24-well ultra-low attachment multiple well plates. Medium was exchanged three times a week.
Live imaging was performed throughout the cultivation periods using the BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan).
For jawbone models, constructs were cultivated in growth medium or mineralisation medium [growth medium supplemented with 10 mM β-glycerophosphate (Sigma-Aldrich, Saint Louis, USA), 10 nM dexamethasone (AppliChem, Darmstadt, Germany) and 284 µM ascorbic acid phosphate (Sigma-Aldrich, Saint Louis, USA)] for up to 28 days in 24-well ultra-low attachment multiple well plates. Medium was exchanged three times a week.
Live imaging was performed throughout the cultivation periods using the BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan).
8
Transmission Electron Microscopy of Tissue Samples
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The specimens were processed for TEM according to a previously published protocol [21 (link)]. In brief, samples were fixed in Ito’s fixative (2.5% glutaraldehyde, 2.5% paraformaldehyde, and 0.3% picric acid) dissolved in phosphate buffered saline (PBS) (pH = 7.3) and embedded in Epon. Semi-thin sagittal sections of 1 μm were cut with a microtome (Ultracut E; Reichert Jung, Vienna, Austria) and subsequently stained with toluidine blue. Sections were viewed with an epifluorescence microscope (Aristoplan; Ernst Leitz, Wetzlar, Germany) and photographed (Keyence Biorevo BZ9000 microscope). Ultrathin sections were stained with uranyl acetate and lead citrate and viewed with a transmission electron microscope (EM109; Carl Zeiss Meditec GmbH, Oberkochen, Germany).
9
Histological and Immunohistochemical Analysis
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The entire fish body was fixed in Davidson’s solution (31% ethanol, 8% formaldehyde, 11.5% acetic acid), embedded in paraffin, serially sectioned, and stained with hematoxylin-eosin for histological analysis. For immunohistochemistry, serial sections with a thickness of 4 μm were stained with the use of a Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA, USA). The sections were thus stained with antibodies to pAKT (#4060; Cell Signaling Technology, Danvers, MA, USA), to pGSK-3β (#9323, Cell Signaling Technology), to CD31 (Ab28364; Abcam, Cambridge, UK), and to Proliferating cell nuclear antigen (NCL-L-PCNA, Leica Biosystems, Newcastle, UK), and they were counterstained with hematoxylin to visualize cell nuclei. Images were captured with the use of a Biorevo BZ-9000 microscope (Keyence, Osaka, Japan).
10
Microscopic Imaging of Live Fish
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Fish were photographed with a Leica DFC300FX camera, and images were processed with Leica Application Suite version 2.7.1.R1 software (Leica Microsystems, Wetzlar, Germany). Moving images were captured with a Biorevo BZ-9000 microscope (Keyence).
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