The right eyes were removed and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. Retinas were flat-mounted on slides, then covered with a mounting medium (Vectashild; Vector Laboratories, Burlingame, CA). For each slide, 3 areas in each quadrant (0.0988 mm2/area, 12 areas in total) were photographed using a fluorescence microscope (BZ-9000; KEYENCE, Osaka, Japan). Each image was obtained using Z-stacking and was stored. Cell counts were performed by image analysis (BZ-9000 software; KEYENCE, Osaka, Japan). Both the number of fluorogold-positive and fluorogold plus Cherry fluorescence double-positive RGCs were used to estimate the transduction efficiency of mVChR1. Furthermore, both the number of fluorogold-positive and fluorogold, Cherry, and Venus fluorescence triple-positive RGCs were used to estimate the percentage of RGCs double-positive for Cherry and Venus fluorescence.
Bz 9000
Manufactured by Keyence
Sourced in Japan, Germany, United States
The BZ-9000 is a digital microscope system designed for comprehensive observation and analysis of samples. It captures high-resolution images and videos of specimens, enabling detailed examination and documentation.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (31 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (24 mentions)
Bz 2 analyzer software, by Keyence (18 mentions)
Lsm 710, by Zeiss (16 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (16 mentions)
Bz 2 analyzer, by Keyence (13 mentions)
Hoechst 33342, by Dojindo Laboratories (12 mentions)
Oct compound, by Sakura Finetek (11 mentions)
Blocking one, by Nacalai Tesque (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (10 mentions)
Fluoromount, by Diagnostic BioSystems (9 mentions)
Paraformaldehyde (pfa), by Fujifilm (9 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (9 mentions)
Tissue tek oct compound, by Sakura Finetek (8 mentions)
Lsm 700, by Zeiss (8 mentions)
Cm3050, by Leica (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions)
Bz analyzer software, by Keyence (7 mentions)
Hoechst 33342, by Merck Group (7 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
982 protocols using bz 9000
1
Quantifying Retinal Ganglion Cell Transduction
2
Retrograde Labeling and Quantification of RGCs
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After the final recording of VEPs (5000 lx exposure), retrograde labeling of RGCs was performed as previously described [9 (link)]. Labeling was performed by injecting 4 μL of 2% aqueous fluorogold (Fluoro-Gold, Fluorochrome, Englewood, CO, USA) containing 1% dimethylsulfoxide into the superior colliculus using a Hamilton syringe with a 32-gauge needle. Seven days after labeling, rat eyes were fixed overnight at 4 °C in 4% paraformaldehyde prepared in phosphate-buffered saline (PBS; Fujifilm Wako Pure Chemical, Osaka, Japan). After rinsing with PBS, the posterior parts such as the cornea, iris, and lens were removed and the retina was detached from the eye cup. The specimen was flat-mounted on a slide and embedded in a mounting medium (Vectashield, Funakoshi, Tokyo, Japan). The flat-mounted retinas were observed and photographed using a fluorescence microscope (Keyence, BZ-9000, Osaka, Japan). Three areas in each quadrant of a whole mounted retina, totaling 12 areas, were photographed with an interval of z-axis at 0.3 μm, with a total of 33 slices. To calculate the number of RGCs, a single image was created from 33 images using the extended depth of field algorithm (full focus) in BZ-9000 software (Keyence, Tokyo).
3
Quantifying Grafted Dopaminergic Neurites in Striatum
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The brains were cut at 50 μm thickness, and the striatum containing a graft was divided into six sets of 12 slices. The number of neurons positive for TH or Girk2 in a graft were counted manually through all graft images of each rat captured by a fluorescence microscope (BZ-9000; Keyence). Graft volume (mm3) and graft surface area (mm2) were calculated by measuring the GFP-positive area of the graft. Neurites double-positive for GFP and Girk2 were derived from the grafted cells (Fig. 3 C). We adopted Girk2 to quantify neurites because of its strong contrast and excellent quantification compared to GFP. Girk2-positive neurites extending from grafted dopaminergic neurons were traced manually and calculated into total neurite length (mm) by using the BZ-9000 Keyence work station. To examine which direction the neurites extended from the graft, the striatum was divided into four areas around the graft: dorsolateral, ventrolateral, dorsomedial, and ventromedial striatum, and total neurite length was calculated and quantified in each area. In addition, to examine how far the neurites from the graft edge reached in the direction of the dorsolateral striatum, contour lines were drawn from the graft edge to a maximum of 500 μm in 100 μm increments, and the number of axons passing through those contours was counted. Imaging analyses were performed with the software Photoshop CC 2018.
4
Quantifying Cellular Uptake and Vascular Remodeling
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To verify cellular uptake of the PLGA NPs (Figure 1, A), vein grafts were excised and soaked in 0.5 mg/mL of FITC-loaded PLGA NP solution or PBS for 30 minutes. Fluorescence was detected and visualized from the vein grafts using an all-in-one fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).
The harvested vein grafts were immersed in 4% paraformaldehyde for 48 hours. Four sections were obtained from each vein graft and stained with hematoxylin and eosin and Elastica Van Gieson. The neointima was defined as the area from the inner surface to the internal elastic lamina. The cross-sectional intimal and medial thicknesses were equiangularly measured at 8 views per section, and the average of these 8 values was used as intimal and medial thicknesses, respectively. The cross-sectional intima/media thickness ratio was also calculated. Each measurement was repeated 3 times. All images were acquired on an all-in-one microscope (BZ-9000; Keyence). Measurements were made using ImageJ software (version 1.50i, National Institutes of Health, Bethesda, Md). 25
The harvested vein grafts were immersed in 4% paraformaldehyde for 48 hours. Four sections were obtained from each vein graft and stained with hematoxylin and eosin and Elastica Van Gieson. The neointima was defined as the area from the inner surface to the internal elastic lamina. The cross-sectional intimal and medial thicknesses were equiangularly measured at 8 views per section, and the average of these 8 values was used as intimal and medial thicknesses, respectively. The cross-sectional intima/media thickness ratio was also calculated. Each measurement was repeated 3 times. All images were acquired on an all-in-one microscope (BZ-9000; Keyence). Measurements were made using ImageJ software (version 1.50i, National Institutes of Health, Bethesda, Md). 25
5
Cardiac Fibrosis and Autophagy Analysis
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Mouse hearts were isolated, and 4%-PFA xed left ventricles were sliced at 7 µm and stained using a Hematoxylin-Eosin Stain Kit and Picro-Sirius Red Stain Kit (COSMO BIO, Japan). Fibrosis was imaged using a BZ-9000 microscope, and brotic areas were quanti ed using the automated calculation software of the BZ-9000 (KEYENCE, Japan). Transduction of green/red uorescent protein-LC3 (GFP-mRFP-LC3) (COSMO BIO) was performed by lipofection into left ventricular sections. The number of green uorescent dots, representing autophagosomes, were counted with the BZ-9000 microscope.
6
Characterization of Peyer's Patches and Intestinal Histology
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Peyer's patches isolated from the mice were embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan) and cut into 6-μm sections by using a cryostat. Sections were fixed in 100% acetone for 1 min at 4°C. To prevent non-specific binding, sections were treated with 2% fetal calf serum in PBS for 30 min at room temperature. Sections were washed with PBS and incubated with biotinylated-CTB, -C-CPE, or -CTB–C-CPE at 4°C overnight. After washing with PBS, sections were stained with Alexa Fluor 546-conjugated streptavidin for 30 min at room temperature. After washing with PBS, sections were stained with 4′,6-diamidino-2-phenylindole (DAPI). The sections were then washed with PBS, mounted in Fluoromount (Diagnostic BioSystems, California, USA), and observed under a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).
Surgically constructed intestinal loop was fixed in 4% paraformaldehyde, embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek Japan), and cut into 6-μm sections by using a cryostat. Sections were stained with hematoxylin for 10 min and eosin for 3 min. The sections were then fixed in ethanol and xylene, mounted in Permount (Thermo Fisher Scientific), and observed under an optical microscope (BZ-9000; Keyence).
Surgically constructed intestinal loop was fixed in 4% paraformaldehyde, embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek Japan), and cut into 6-μm sections by using a cryostat. Sections were stained with hematoxylin for 10 min and eosin for 3 min. The sections were then fixed in ethanol and xylene, mounted in Permount (Thermo Fisher Scientific), and observed under an optical microscope (BZ-9000; Keyence).
7
Immunostaining for Syndecan-1 and BMMNC Tracking
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The sections were blocked by 20% Block Ace (Dainippon Sumitomo Pharma Co., Osaka, Japan) in 0.1 M PB with 0.005% saponin (Sigma-Aldrich Co., St. Louis, MO) and incubated with the primary antibody, rabbit monoclonal anti-syndecan-1 antibody (EPR6454; abcam), followed by the secondary antibody, goat anti-rabbit IgG antibody (Goat Anti-Rabbit IgG H&L [HRP]; abcam), overnight at 4 °C. The antibodies were dissolved in 0.1 M PB containing 5% Block Ace and 0.005% saponin. The sections were washed with PBS after each reaction. Finally, they were mounted in 0.1 M PB-glycerin (1:1) solution and observed under a fluorescent microscope (BZ-9000; Keyence Co.). For negative control, nonimmune serum was substituted for the primary antibody.
To investigate the distribution and engrafting of transplanted BMMNCs in the host tissues, we also injected GFP-derived BMMNCs into the heatstroke rats (GFP-BMMNCs group, n = 9). Sections of lung, kidney, and spleen were observed under a fluorescent microscope (BZ-9000; Keyence Co.) at 1 day (n = 3), 1 week (n = 3), and 2 weeks (n = 3) after heat stress.
To investigate the distribution and engrafting of transplanted BMMNCs in the host tissues, we also injected GFP-derived BMMNCs into the heatstroke rats (GFP-BMMNCs group, n = 9). Sections of lung, kidney, and spleen were observed under a fluorescent microscope (BZ-9000; Keyence Co.) at 1 day (n = 3), 1 week (n = 3), and 2 weeks (n = 3) after heat stress.
8
Immunostaining of Pluripotent Stem Cells
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The immunostaining was performed using the following primary antibodies and reagents: anti-OCT4 (sc-5279, Santa Cruz, CA, USA), anti-NANOG (RCAB0003P, ReproCELL, Yokohama, Japan), anti-SSEA 4 (MAB4304, Millipore), anti-Tra1-60 (MAB4360, Millipore, MA, USA), anti-human smooth muscle actin monoclonal antibody (Agilent Technologies, CA, USA), goat anti-human SOX17 polyclonal antibody (R&D Systems, MN, USA), rabbit anti-Nestin polyclonal antibody (Sigma), or Prolyl 4-hydroxylase beta (PH4B) (Acris Antibodies GmbH, Herford, Germany), together with 4′,6′-diamidino-2-phenylindole (DAPI; ThermoFisher SCIENTIFIC, MA, USA) [13] (link). Signal was detected using a conventional fluorescence laser microscope (BZ-9000, KEYENCE, Osaka, Japan) equipped with a color charge-coupled device camera (BZ-9000, KEYENCE).
9
Quantifying Microglial Activation in Imaging
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Twenty Z-stack images were acquired at a thickness of 40 μm separated by 2-μm intervals and converted to one Z-projection image. The images for cell counting and cell body area measurements of Iba1-positive cells were examined using a fluorescence microscope (model BZ-9000, Keyence, Osaka, Japan). We counted the Hoechst 33258-stained nuclei of Iba1-positive microglia using the Cell Counter plugin of ImageJ 1.44 (NIMH; Bethesda, MD, USA). The body area of Iba1-positive cells was examined using a fluorescence microscope (model BZ-9000, Keyence, Osaka, Japan). The average cell number and body area of four images were used as one data.
10
Histological Analysis of Organ Remodeling
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Histological analysis was performed as described previously.17 , 18 The heart, aorta, and kidney were fixed with 4% PFA and embedded in paraffin. Sections (4‐μm thick) were stained with hematoxylin and eosin, periodic acid–Schiff, Masson's trichrome, and Elastica van Gieson. For analysis of renal structures, glomerular sclerosis was semiquantitatively evaluated and expressed as the glomerular sclerosis index, as described previously.21 Briefly, 30 glomeruli per sample were graded as 0, 1, 2, 3, or 4, indicating absent, <25, 25 to 50, 51 to 75, or >75% sclerosis, respectively. Cardiac and renal fibrotic areas and aortic medial thickness were measured digitally using a fluorescence microscope (BZ‐9000; Keyence, Osaka, Japan). Immunohistochemistry was performed as described previously.18 , 22 Sections were incubated with either an anti‐SIRT1 antibody (1:50; 07‐131; Millipore, Billerica, MA) or anti‐4‐hydroxy‐2‐nonenal (4‐HNE) antibody (1:100; MHN‐100P; JaICA, Haruoka, Japan). The 4‐HNE‐stained area was measured digitally using a fluorescence microscope (BZ‐9000; Keyence).
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