Half of the lungs of each mouse was isolated and fixed in 4% paraformaldehyde for 5–7 h at RT, dehydrated in ascending isopropanol dilutions and embedded in paraffin. Paraffin sections of 4 µm were analyzed. Lung specimens were rehydrated and heat-mediated antigen retrieval was performed (10mM citric acid buffer pH 6.0, 20 min). Afterwards paraffin sections were blocked with 10% fetal bovine serum containing 0.1 % Triton-X-100 for 30 min. Antigen-specific staining of human c-Raf-1 and influenza virus NP were performed by incubation with appropriate primary antibodies (rabbit anti-human c-Raf [SP-63], 1:500; goat anti-influenza NP [G105] 1:2500, kind gift of Dr. Robert Webster, Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN) for 1h at RT followed by species-specific secondary antibody incubation for 30 min. The Vectastain ABC-AP Kit (Vector Laboratories, Cat. No.: AK-5000) was used for visualization of the stained proteins as described in the manufacturer´s protocol. Three different sections per mouse lung (approx. 250 µm apart from each other) were quantified from at least 5 mice per each time point. The area of the Raf-positive tumor foci was then measured and expressed in relation to the total section area of the specimen. All analyses were quantified in a blinded manner via the Keyence BZ Analyzer (Keyence).
Bz analyzer
Manufactured by Keyence
Sourced in Japan
The BZ analyzer is a compact and user-friendly device designed for the measurement of oxygen concentration. It utilizes an electrochemical sensor to provide accurate and reliable oxygen readings. The BZ analyzer is suitable for a variety of applications where the monitoring of oxygen levels is required.
Automatically generated - may contain errors
Lab products found in correlation
Bz 9000, by Keyence (6 mentions)
Bz x710, by Keyence (5 mentions)
Biozero, by Keyence (3 mentions)
Bz 8000, by Keyence (2 mentions)
Bz x700 microscope, by Keyence (2 mentions)
Mouse anti glucagon, by Abcam (2 mentions)
Guinea pig anti insulin, by Merck Group (2 mentions)
Bz x700, by Keyence (2 mentions)
Hybrid cell count mode, by Keyence (2 mentions)
Alexa 488 or alexa 594 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Vectastain abc ap kit, by Vector Laboratories (1 mentions)
Cd66b clone g10f5, by BD (1 mentions)
Cd8 clone sp16, by Abcam (1 mentions)
Histogreen, by Cosmo Bio (1 mentions)
Hoechst 33342, by Dojindo Laboratories (1 mentions)
Fv300 laser confocal microscope, by Olympus (1 mentions)
Digoxigenin rna labeling kit, by Roche (1 mentions)
Hoechst dye, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Alkaline phosphatase, by Roche (1 mentions)
42 protocols using bz analyzer
1
Quantification of Raf-Positive Lung Tumors
2
Multimodal Immunohistochemistry Analysis
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CRC tissues were fixed using neutral-buffered formalin and embedded in paraffin. The sections were stained with Perls’ reagent and developed using DAB, as previously described (42 (link)). After iron staining, the slides were subsequently incubated with primary antibodies against CD8 (clone SP16, ab9829; Abcam), CD66b (clone G10F5, 555723; BD Pharmingen), cytokeratin 20 (clone Ks20.8, 413491; Nichirei), and IBA1 (polyclonal, 019-19741; Wako) overnight at 4°C. The sections were visualized using HistoGreen (E109; Cosmo Bio) and counterstained with Mayer hematoxylin. CCL8 (clone 1.1_2D4-1A3, LS-B8198; LSBio) staining was conducted using DAB, followed by costaining with IBA1 using HistoGreen. Images were obtained with a KEYENCE BZ-X800 all-in-one microscope (KEYENCE). Quantification was performed using the KEYENCE BZ analyzer.
3
Quantifying PDL Cell Expression
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The middle one-third buccal aspect of the PDL on the distal palatal root was photographed using an optical microscope (Biozero; Keyence). Quantitative images were measured using image analysis software (BZ analyzer; Keyence). The number of bFGF- and VEGF-positive PDL cells was counted in a rectangular area (300×400 µm) (Fig. 2B ). Three representative sections from each of the five samples of all groups were measured in a blinded manner.
4
Histological Analysis of Wound Healing
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The 8 µm thick sections were fixed with 10% formalin and stained with H-E according to a standard protocol. After staining, the sections were photographed at 40× magnification using a light microscope (BZ-9000; KEYENCE, Osaka, Japan) and combined to create a picture of the whole wound using image analysis software (BZ-analyzer; KEYENCE). The epithelial gaps were measured photogrammetrically.
5
Quantitative Analysis of Tumor Vasculature
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For analyses of the microvessel density (MVD), the total areas of CD31, vWF-positive capillaries, and venules were assessed by scanning tumor sections under × 40 magnification and counting in 10 random fields under × 600 magnification
[44 (link),45 (link)]. Pericytes were identified by scanning tumor sections under × 1000 magnification and counting in 3 random fields under × 1000 magnification
[46 (link)]. These data were analyzed using a BZ-Analyzer (Keyence, Osaka, Japan).
[44 (link),45 (link)]. Pericytes were identified by scanning tumor sections under × 1000 magnification and counting in 3 random fields under × 1000 magnification
[46 (link)]. These data were analyzed using a BZ-Analyzer (Keyence, Osaka, Japan).
6
Lu-Fc Binding Assay on Mouse Kidney Sections
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The Lu-Fc binding assay was performed as described previously [19 (link)]. Briefly, Lu-Fc was diluted with PBS(-) containing 10 μg/ml of antibody or 5–12 × 1011 pfu/ml of phage antibodies and was then placed on adult mouse kidney sections. The α5-containing laminin, the specific ligand of Lu, is enriched in the basement membrane of the kidney. Therefore, we could conveniently perform the binding assay for Lu on the tissue without purifying the α5-containing laminins. Animal studies were approved by the Animal Research Committee of Tokyo University of Pharmacy and Life Sciences (P15-10). Mice were sacrificed under deep anesthesia with Pentobarbital. Bound Lu-Fc was detected with anti-human IgG antibody conjugated to Alexa488 (ThermoFisher Scientific). The anti-laminin α5 polyclonal antibody was used for counter staining. The fluorescence intensities of the bound recombinant proteins were normalized with those of laminin α5 staining in the same areas and quantified using a BZ-analyzer (Keyence, Osaka, Japan).
7
Anchorage-Independent Spheroid Formation
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The ability of cells to grow in an anchorage-independent manner was assessed to evaluate the spheroid formation. Cells were seeded in 10% FBS containing RPMI soft agar at a density of 5 × 103 cells in 24-well plate. On day 7 after seeding, spheroid formation was assessed by staining with Hoechst33342 (Dojindo Laboratories, Kumamoto, Japan). The number and size of spheroid were measured by BZ analyzer (KEYENCE, Osaka, Japan).
8
Localization of SLC18B1 Protein in Rat and Mouse Brains
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Indirect immunofluorescence microscopy was performed as described57 . Brain samples were obtained from male Wistar rats or C57BL/6 mice perfused intracardially with 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Cultured cells on poly-l -lysine-coated coverslips were fixed with 4% paraformaldehyde in PBS for 30 minutes. The primary antibody against mouse SLC18B1 protein was diluted 1:200 (v/v) with PBS containing 0.5% (w/v) BSA and the sample was incubated for 1 hour at room temperature. Washing steps and secondary antibody treatment were performed as described57 . The specimens were observed either under an Olympus FV300 confocal laser microscope (Olympus) or BIOZERO (Keyence).
For immunoperoxidase labeling, coronal sections of the mouse brain 30 μm thick were cut on a freezing microtome. Immunoperoxidase staining was performed on free-floating sections according to standard immunoperoxidase protocols as described58 (link). The sections were photographed with BIOZERO (Keyence) and merged to obtain whole brain images using image-joint software BZ-Analyzer (Keyence).
For immunoperoxidase labeling, coronal sections of the mouse brain 30 μm thick were cut on a freezing microtome. Immunoperoxidase staining was performed on free-floating sections according to standard immunoperoxidase protocols as described58 (link). The sections were photographed with BIOZERO (Keyence) and merged to obtain whole brain images using image-joint software BZ-Analyzer (Keyence).
9
Detecting Fibronectin mRNA Expression
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Digoxigenin-11-dUTP single-stranded RNA probes for detecting fibronectin mRNA were prepared using a digoxigenin RNA labeling kit (Roche). The sections were treated with proteinase K and acetic anhydride and overlaid with 150 μl of hybridization solution containing the digoxigenin-labeled fibronectin probe (1 μg/ml). Then, they were denatured at 70°C for 60 minutes and hybridized overnight at 65°C. Hybrids were detected with an anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche). Sections were incubated in 1% bovine serum albumin/PBS for 1 hour before incubation with the primary antibody. Primary antibodies specific for fibronectin (kindly provided by K. Yamada), collagen IV [1 (link)], dentin sialoprotein (DSP) [20 (link)], ameloblastin [20 (link)], laminin β1γ1 (MAB1905; Merck Millipore), and β1 integrin (9EG7: BD Biosciences) were detected using Alexa488- or Alexa594-conjugated secondary antibodies (Invitrogen). Nuclei were stained with Hoechst dye (Sigma-Aldrich) or DAPI (Vector Laboratories). A fluorescence microscope (BZ-8000, KEYENCE) was used for imaging analysis. Images were prepared using a BZ analyzer (KEYENCE) and Adobe Photoshop (Adobe Systems, Inc.).
10
NF-κB p65 Activation Imaging
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Briefly, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Then, cells were stained with anti–NF-κB p65 antibody (8242; Cell Signaling Technology) and Alexa Fluor 488–labeled goat secondary antibody (A-11008; Invitrogen). Images were obtained with the KEYENCE BZ-X800, and quantification was performed using a BZ analyzer (KEYENCE).
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