The mice were transcardially perfused with phosphate-buffered saline (PBS) followed by 4% PFA-PBS solution. The brains were post-fixed in 4% PFA-PBS for overnight at 4 °C and placed in 30% sucrose PBS solution for 2 days. Coronal sections were obtained using a cryostat with a thickness of 40 μm. The sections were analyzed using a fluorescence microscope (KEYENCE BZ-X810) with a 10X, 20X or 40X objective. For AAV2-retro experiments, the sections including the primary cortical sensory area (S1) or the nucleus accumbens (NAc) were imaged. The fluorescence intensity of the cells and the number of cells were automatically measured with a software (KEYENCE Image Analyzer). Since retrograde projections are sparse in the above-mentioned circuits, we quantified GFP signal to evaluate the transduction efficiency as conducted in the other studies [23 (link)–25 (link)]. For AAV1 experiments, fifty consecutive sections including the superior colliculus (SC) were imaged per a mouse. The number of cells was automatically measured with a software (KEYENCE Image Analyzer). During the whole imaging and analysis processing, the information of samples (the original virus or the mutant virus) was blinded to the experimenter.
Image analyzer
Manufactured by Keyence
Sourced in Japan
The Image Analyzer is a device that captures and analyzes digital images. It provides tools for measurement, defect detection, and image processing. The core function of the Image Analyzer is to acquire and process visual data for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Bz x700 fluorescence microscope, by Keyence (1 mentions)
Rhodamine dextran, by Merck Group (1 mentions)
Lucifer yellow, by Merck Group (1 mentions)
Bz x810, by Keyence (1 mentions)
Fitc labeled streptavidin, by Thermo Fisher Scientific (1 mentions)
Biotin conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Biotin conjugated anti rabbit igg and fitc labeled streptavidin, by Thermo Fisher Scientific (1 mentions)
α sma, by Agilent Technologies (1 mentions)
Calcein am, by Dojindo Laboratories (1 mentions)
Bz x800 microscope, by Keyence (1 mentions)
9 protocols using image analyzer
1
Quantification of Sparse Neuronal Projections
2
Quantification of Glutathione S-Transferase Placental Foci
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The glutathione S-transferase placental form (GST-P) was immunohistochemically stained, as previously described [25 (link)]. Averages of GST-P–positive foci that were >80 μm in diameter in the entire liver section were evaluated using an image analyzer (Keyence, Osaka, Japan).
3
Cholangiocyte Organoid Expansion Assay
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EpCAM+ cholangiocytes were cultured in type I collagen gel. Each well of a 24-well plate was coated with 50 μl collagen gel and 10,000 cholangiocytes suspended in 100 μl collagen solution on ice were then plated. After incubation at 37 °C for 30 min, 600 μl of basal medium containing 5 ng/ml EGF and 100 ng/ml HGF was added to each well. Seven days after plating, the medium was replaced with that containing 10 μM ISO. The size of cholangiocyte organoids was assessed before ISO addition and 2 days after ISO addition. Three wells were assessed for each condition and four to five organoids in each well were identified and recorded at culture days 7 and 9 using a Keyence BZ-X700 fluorescence microscope (Osaka, Japan). The luminal areas of those organoids on 2D images were measured using a Keyence Image Analyzer. Each culture was independently repeated four times.
4
Measuring Gap Junction Functionality
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This procedure was performed according to the method described in an earlier report [40 (link)]. Briefly, liver samples were obtained from Wt rats with each treatment (4 rats per group) and treated with Lucifer yellow (Sigma-Aldrich Corp., St. Louis, MO), a stain that can pass through the gap junction channel, and rhodamine-dextran (Sigma-Aldrich Corp.), which does not cross through the channel, to measure gap junction capability. Liver slices were cut to 5 mm-thick and 3 incisions of 1 mm depth were made, followed by the addition of a mixture of fluorescent dyes containing 0.05% Lucifer yellow and 0.05% rhodamine-dextran in PBS into the incisions. After 3 minutes, the slices were washed 3 times with PBS and frozen. Thereafter 7 μm thick frozen sections were made and spread of the dye was measured using an image analyzer (Keyence).
5
Fluorescence Immunohistochemistry for Connexins
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The detailed methods for fluorescence immunohistochemistry employed in this study have been described previously [22 (link)]. Frozen sections were cut to 6 μm thickness and fixed in cold acetone and 10% buffered formalin. A polyclonal rabbit antibody against Cx32 (Thermo Fischer Scientific Inc., Waltham, MA) was used with biotin-conjugated anti-rabbit IgG and TRITC-labeled streptavidin (Thermo Fischer Scientific Inc.) to visualize the endogenous proteins using an image analyzer (Keyence). A monoclonal mouse antibody against Cx26 (Thermo Fischer Scientific Inc.) was used with biotin-conjugated anti-mouse IgG and FITC-labeled streptavidin (Thermo Fischer Scientific Inc.).
6
Hepatic GST-P Immunohistochemical Staining
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Immunohistochemical staining of glutathione S-transferase placental form (GST-P) was performed in accordance with our previous report (Naiki-Ito et al. 2012 (link)). The average number and area of GST-P-positive foci > 80 μm in diameter in the total area of the liver section were measured with an image analyzer (Keyence, Osaka, Japan).
7
Immunofluorescence Staining of LLGL2 and LAT1
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The detailed methods for immunofluorescence staining employed in this study have been described previously27 (link). Frozen sections were cut to 4-μm thickness and fixed in cold acetone and 10% buffered formalin. A mouse monoclonal anti-LLGL2 antibody (Abnova Corporation, Taipei, Taiwan) was used with biotin-conjugated anti-mouse IgG and TRITC-labeled streptavidin (Thermo Fisher Scientific) to visualize the endogenous proteins using an image analyzer (Keyence). A rabbit monoclonal anti-SLC7A5/LAT1 antibody (Abcam, Cambridge, UK) was used with biotin-conjugated anti-rabbit IgG and FITC-labeled streptavidin (Thermo Fisher Scientific).
8
Histological Evaluation of Steatohepatitis
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The degree of steatohepatitis was evaluated as previously described in detail (Sagawa et al. 2015 (link)). Briefly, formalin-fixed liver sections were stained with hematoxylin and eosin (H&E) or Azan, and were also used for immunohistochemical measurement of α-smooth muscle actin (α-SMA, Dako, Tokyo, Japan). The positive areas of Azan and α-SMA immunostaining were measured with an image analyzer (Keyence, Osaka, Japan). The progression of steatohepatitis was analyzed using a non-alcoholic fatty liver disease activity score (NAS): the score represents a sum of 3 subscores; namely, severity of steatosis (0–3), lobular inflammation (0–2), and hepatocyte ballooning (0–3). NAS and scores for fibrosis (0–4) were determined by three experienced pathologists (AN, HK, and ST), according to the method described by Kleiner et al. (2005 (link)).
9
Islet Cell Viability Assessment
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To assess the viability of islet cells, Calcein-AM and propidium iodide (PI; Dojindo Laboratories, Kumamoto, Japan) were used as viability stains. Fresh solutions were prepared by diluting the stock in PBS to yield a final concentration of 1 μg/mL Calcein-AM and 10 μg/mL PI. The isolated islets were cultured for 24 h in the presence of trametinib (final concentration, 1 nmol/L–1 μM) or solvent (DMSO), and then incubated with the staining solution for 10 min at 37°C. The islets were then washed and assessed using fluorescence microscopy (BZ-X800 microscope; KEYENCE, Japan). Viable cells convert Calcein-AM to a green fluorescent product. Dead cells are permeable to PI, which is observed as red fluorescence. The viability of each islet was measured as a percentage of the total Calcein-AM positive area, subtracting the PI positive area from total Calcein-AM and PI positive areas. The areas were calculated using an image analyzer (KEYENCE, Japan).
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