Isolated pancreata were immersion fixed in 4% paraformaldehyde. The tissue was routinely processed for paraffin embedding, and 5-mm sections mounted on glass slides were immunostained with goat anti-rabbit insulin antibody (diluted 1:1,000; Proteintech Group Inc., Rosemont, IL). We analyzed two sections of each pancreas that were 50 mm apart. b-Cell area was calculated using a BZ-II analyzer (Keyence Co., Osaka, Japan). b-Cell mass was estimated for each animal by determining the b-cell area as a proportion of total pancreatic area per animal and multiplying this proportion by the pancreas weight. For immunofluorescence, tissue sections were incubated overnight at 4°C with the primary antibodies listed in Supplementary Table 1. After rinsing with PBS, tissues were incubated with secondary antibodies for 60 min (diluted 1:200) using previously described procedures (25) (Supplementary Table 1). Immunofluorescence images were acquired using a BZ-II analyzer (Keyence Co.) according to the manufacturer's instructions.
Bz 2 analyzer
Manufactured by Keyence
Sourced in Japan, Germany, United States
The BZ-II Analyzer is a device designed for analyzing various samples. It provides accurate measurement and data collection capabilities.
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Lab products found in correlation
Biorevo bz 9000, by Keyence (20 mentions)
Bz 9000, by Keyence (13 mentions)
Bz 9000 fluorescence microscope, by Keyence (6 mentions)
Bz 9000 microscope, by Keyence (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Bz x700, by Keyence (4 mentions)
Bz 2 viewer, by Keyence (4 mentions)
Bz 9000 all in one fluorescence microscope, by Keyence (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Fujifilm (2 mentions)
Fluorescence microscope, by Keyence (2 mentions)
Bz 8000 microscope, by Keyence (2 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
Biorevo bz 9000 microscope, by Keyence (2 mentions)
Axio observer, by Zeiss (2 mentions)
Bz 8100, by Keyence (2 mentions)
Fitc dextran, by Merck Group (2 mentions)
132 protocols using bz 2 analyzer
1
Quantification of Pancreatic β-Cell Mass
2
Quantitative Pancreatic Islet Analysis
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Isolated pancreata were immersion-fixed in 4% paraformaldehyde at 4 °C overnight, then paraffin-embedded, and 5-µm sections were mounted on glass slides. For immunofluorescence, tissue sections were incubated at 4 °C overnight with the primary antibodies listed in Supplementary Table S2 . After rinsing with PBS, the sections were incubated with secondary antibodies for 30 min. Immunofluorescence images were acquired using a BZ-II analyzer (Keyence, Osaka, Japan). The mitochondrial area was quantified using a BZ-II analyzer (Keyence, Osaka, Japan). The mitochondrial area/beta-cells ratio was expressed as the Tom 20 positive area inside the insulin positive area per islet divided by the number of beta-cells. The Nkx6.1 positive cells/beta-cells ratio was expressed as the number of cells with strong nucleic Nkx6.1 staining among the insulin positive cells per islet divided by the number of beta-cells.
3
Insulin-Positive Pancreatic β-Cell Mass
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Pancreata were fixed with 10% formalin, minced with a razor, and embedded in paraffin. The sections were immunostained with monoclonal anti-insulin antibody (I2018, Sigma). Immunoreactivity was visualized by incubation with a substrate solution containing 3,3′-diaminobenzidine tetrahydrochloride (DAB). The tissue samples were sectioned at an interval of 100 µm and the ratio of insulin-positive areas to total pancreatic tissues was determined using a microscope, the BIOREVO BZ-9000 (Keyence, Osaka, Japan), and a BZ-2 Analyzer (Keyence). β-Cell mass was calculated by multiplying the ratio of insulin-positive areas by pancreatic weight.
4
Articular Disc and Retrodiscal Tissue Analysis
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The thickness of the anterior, central, and posterior parts of the articular disc were measured in the digital images of the hematoxylin-eosin stained specimens (n = 5 from each group) using an image analysis and measurement software (BZ-2 Analyzer, Keyence) (Fig. 1).
Ten capillaries of the retrodiscal tissue were randomly selected from the digital images of the microvascular corrosion cast specimens (n = 5 from each group). The capillaries of the retrodiscal tissue were measured using a measurement software (Image-Pro Plus ® 5.1J, Nippon Roper, Tokyo, Japan).
All measurements were calculated and represented as means ± standard deviations, and Student's t-test was used to determine statistical significant differences between the 2 groups. Differences were considered significant when p < 0.01.
Ten capillaries of the retrodiscal tissue were randomly selected from the digital images of the microvascular corrosion cast specimens (n = 5 from each group). The capillaries of the retrodiscal tissue were measured using a measurement software (Image-Pro Plus ® 5.1J, Nippon Roper, Tokyo, Japan).
All measurements were calculated and represented as means ± standard deviations, and Student's t-test was used to determine statistical significant differences between the 2 groups. Differences were considered significant when p < 0.01.
5
Quantifying Synovial GRK5 Expression
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GRK5 localisation in each synovial layer was systematically assessed by counting the number of positive cells. The frequency of positive cells was expressed as a percentage relative to the total number of cells counted in each layer with the BZ-II Analyzer software program (Keyence).
6
Quantifying Fluorescent Cell Counts
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The obtained fluorescence images on BZ Viewer® were analyzed by the original software BZ-II analyzer®, developed by Keyence Co, Ltd., Osaka, Japan. In this study, we counted the number of cells emitting red fluorescence under the Cy5 filter in ×100 images.
7
Immunofluorescence Imaging of Liver Sections
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Liver sections (10 μm) were cut with a Leica CM1950 Clinical Cryostat (Leica, Wetzlar, Germany), washed with PBS three times (3–5 minutes each time), fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes, and rinsed in PBS three times at room temperature. Samples were then blocked in 5% NFDM in PBST for 1 hour at room temperature. Rabbit anti-GFP (1:200, PA1-980A; ThermoScientific Pierce Products) was used as primary antibody in 5% NFDM in PBST and samples were incubated overnight at 4 °C. Sections were washed three times in PBS and incubated with the secondary antibody Alexa Fluor 594 (1:200, R37117; Thermo Scientific Pierce Products) and Hoechst 33342 (1:2000, H3570; Bioprobes) for 1 hour at room temperature. After incubation, slides were washed three times and mounted using FluorSave Reagent (Calbiochem). Individual images were acquired using the HS All-in-one Fluorescence Microscope BZ-9000E (Keyence, Osaka, Japan) with a 20x objective and the BZ-II Analyzer (Keyence, Osaka, Japan).
8
Multicolor Immunofluorescence Staining of Mice Lymphoid Organs
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Mice frozen lymphoid organ sections were fixed with 4% PFA for 10 min at room temperature and blocked with 5% FBS plus 5% rat serum for 1 h at room temperature. Then the section was stained with either 2.5 µg/ml of CD8 (53–6.7) Alexa Fluor 594, 2.5 µg/ml of CD4 (GK1.5) Alexa Fluor 647, and 2.5 µg/ml of B220 (RA3-6B2) Alexa Fluor 488, or 2.5 µg/ml of F4/80 (BM8) Alexa Fluor 647 and 2.5 µg/ml of CD11c (N418) Alexa Fluor 488 overnight at 4°C. All antibodies were purchased from BioLegend.
Images were acquired using a Keyence BZX-700 all-in-one fluorescence microscope with CFI Plan Apochromat λ4× fluorescent objective (Nikon Plan Apochromat; 0.2 NA) or CFI Plan Apochromat λ10× fluorescent objective (Nikon Plan Apochromat; 0.45 NA), which was operated with a 2/3-in, 2.83 million pixel monochrome charge-coupled device colorized with a liquid chromatography filter at 25°C. Fluorochromes used for these analyses were Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 488, and/or DAPI. Images were processed and analyzed by a BZ-II Analyzer (Keyence) and Photoshop Element 10 software (Adobe). Background was reduced using brightness and contrast adjustments applied to the whole image.
Images were acquired using a Keyence BZX-700 all-in-one fluorescence microscope with CFI Plan Apochromat λ4× fluorescent objective (Nikon Plan Apochromat; 0.2 NA) or CFI Plan Apochromat λ10× fluorescent objective (Nikon Plan Apochromat; 0.45 NA), which was operated with a 2/3-in, 2.83 million pixel monochrome charge-coupled device colorized with a liquid chromatography filter at 25°C. Fluorochromes used for these analyses were Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 488, and/or DAPI. Images were processed and analyzed by a BZ-II Analyzer (Keyence) and Photoshop Element 10 software (Adobe). Background was reduced using brightness and contrast adjustments applied to the whole image.
9
Quantitative Analysis of Grafted Cells
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Randomly selected high-power fields corresponding to the peri-impact region were used to count surviving grafted cells and inflammatory cells in this region. Briefly, 40 μm cryostat-sectioned tissues were examined at 4× magnification and digitized using Axio Vision (Zeiss) and BZ-II Analyzer (Keyence). Brain sections were blind-coded and Abercrombie's formula was used to calculate the total number of immunopositive cells 32 (link), 33 (link).
For each labeling treatment, six coronal sections of the brain and eight coronal sections of the spleen were collected from each animal. From each section, approximately 4 to 6 images at 20× magnification were taken using a confocal microscope (Zeiss) and analyzed with ImageJ (National Institutes of Health, Bethesda, MD). All photomicrographs were converted to gray scale. Background was selected from blank control images and subsequently used to subtract the background from all images. The same threshold was used for all images. Thereafter, the labeling intensity of each section was quantified as the average optical density readings of 4 to 6 randomly selected areas within that section. The final labeling intensity of each group was expressed as the average of each labeling intensity per section.
For each labeling treatment, six coronal sections of the brain and eight coronal sections of the spleen were collected from each animal. From each section, approximately 4 to 6 images at 20× magnification were taken using a confocal microscope (Zeiss) and analyzed with ImageJ (National Institutes of Health, Bethesda, MD). All photomicrographs were converted to gray scale. Background was selected from blank control images and subsequently used to subtract the background from all images. The same threshold was used for all images. Thereafter, the labeling intensity of each section was quantified as the average optical density readings of 4 to 6 randomly selected areas within that section. The final labeling intensity of each group was expressed as the average of each labeling intensity per section.
10
Masson's Trichrome Staining of Paraffin-Embedded Heart
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The paraffin-embedded heart sections were stained with Masson's trichrome by using a trichrome stain kit (ScyTek Laboratories, Inc, Logan, UT) in accordance with the manufacturer's instructions. The stained sections were observed under fluorescence microscopy (BZ-9000, Keyence, Osaka, Japan), and the infarct and left ventricular areas were assessed using a BZ-II analyzer (Keyence).
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