Approximately 5000 hMSCs were plated onto cover slips. The cells were incubated with 160 μM H2O2 for 24 h after being cultivated for 24 h. Cells were fixed with 4% paraformaldehyde solution for 20 min and then rinsed with PBS. For permeabilization, cells were incubated with 1% Triton X-100 PBS solution for 20 min at RT. After washing with PBS, unspecific binding sites were blocked with blocking buffer (5% goat serum in PBS) for 45 min at RT. Then, cells were incubated with primary antibody anti-Dnmt3a (10 μg/mL) overnight at 4°C. After washing three times with PBS, cells were incubated with secondary antibody solution (Alexa Fluor® 546 goat anti-mouse) diluted 1 : 500 in PBS solution for 1 h in the dark at RT. Nuclei were counterstained by incubation with DAPI (1 μg/mL) for 5 min at RT. After washing three times to remove unbound DAPI, the stained cells were mounted with mounting medium. Images of the staining were taken with a fluorescence microscope (VB-6000, Keyence, Japan) under standardized conditions and analyzed with ImageJ 1.45s software.
Vb 6000
Manufactured by Keyence
Sourced in Japan
The VB-6000 is a high-resolution microscope system designed for precise observation and measurement of small-scale samples. It features a compact and modular design, allowing for easy integration into various lab environments.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Eclipse e400, by Nikon (1 mentions)
Proteinase k, by Nacalai Tesque (1 mentions)
Directpcr lysis reagent, by Viagen Biotech (1 mentions)
Lsm 710 laser scanning microscope, by Zeiss (1 mentions)
Leica af6500, by Leica Microsystems (1 mentions)
Rabbit anti somatostatin, by Abcam (1 mentions)
Goat anti pancreatic polypeptide, by Abcam (1 mentions)
Rabbit anti ki67, by Abcam (1 mentions)
Rabbit anti vegf, by Abcam (1 mentions)
7 protocols using vb 6000
1
Immunofluorescence Staining of hMSCs
2
Histological and Immunohistochemical Assessment of Liver
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For histological evaluations, liver tissues were fixed in 10% buffered formalin and embedded in paraffin, and Hematoxylin-Eosin (H-E) staining was performed. Hepatic lipid accumulation was measured morphometrically.
The expression and localization of tissue 4-HNE in the liver was detected by immunohistochemical staining as previously described elsewhere.(18 (link)) Briefly, deparaffinized tissue sections were incubated with a monoclonal anti-4-HNE antibody and secondary biotinylated antimouse IgG, and the specific binding was visualized with the abidin-biotin complex solution followed by incubation with a 3,3-diaminobenzidine tetrahydrochloride solution using Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Specimens for histology and immunohistochemistry were observed under an optical microscope (PH-2; Olympus, Tokyo, Japan) equipped with a digital microscope camera (VB6000; Keyence, Osaka, Japan).
The M30 cytodeath assay, which stains a caspase cleavage product of cytokeratin (ccCK) 18, was performed following the manufacturer's instructions. Briefly, deparaffinized tissue sections were incubated with a monoclonal anti-M30 antibody and secondary biotinylated antimouse IgG, and specific binding was visualized as above.
The expression and localization of tissue 4-HNE in the liver was detected by immunohistochemical staining as previously described elsewhere.(18 (link)) Briefly, deparaffinized tissue sections were incubated with a monoclonal anti-4-HNE antibody and secondary biotinylated antimouse IgG, and the specific binding was visualized with the abidin-biotin complex solution followed by incubation with a 3,3-diaminobenzidine tetrahydrochloride solution using Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Specimens for histology and immunohistochemistry were observed under an optical microscope (PH-2; Olympus, Tokyo, Japan) equipped with a digital microscope camera (VB6000; Keyence, Osaka, Japan).
The M30 cytodeath assay, which stains a caspase cleavage product of cytokeratin (ccCK) 18, was performed following the manufacturer's instructions. Briefly, deparaffinized tissue sections were incubated with a monoclonal anti-M30 antibody and secondary biotinylated antimouse IgG, and specific binding was visualized as above.
3
Meibomian Gland Orifice Imaging
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Formalin-fixed eyelids, skin, and subcutaneous tissues were manually removed using fine forceps. Specimens were then bathed overnight in 15%, and then 30% sucrose in PBS (Gibco by Life Technologies, Carlsbad, CA, USA). This process made the connective tissues, including palpebral tissue, transparent, while keeping the lipid-rich MGs opaque. Photographs of the MGs were taken using a dissecting microscope with a transmitted light source and a digital camera (VB-6000, Keyence, Osaka, Japan). Digital MG photographs were then converted into grayscale images using Adobe Photoshop CS4 (Adobe, San Jose, CA, USA). Fulgurated MG orifices appeared black in these images, and the MG orifice surface area was measured using ImageJ 1.49 software (National Institutes of Health, Bethesda, MD, USA).
4
Detecting Apoptosis with TUNEL Assay
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To detect apoptosis directly, the cells were analyzed with TUNEL (In Situ Cell Death Detection Kit, TMR red; Roche Diagnostics GmbH, Mannheim, Germany). Briefly, the cells were placed on glass slides and dried. They were then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature, washed in PBS, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4°C. TdT labeling was performed with the commercially available kit, according to the manufacturer’s protocol. The apoptotic cells were directly detectable by their red color with fluorescence microscopy (Eclipse E400, Nikon Inc., Melville, NY). The images were captured with a CCD camera and processed with a digital fluorescence microscope (VB-6000, Keyence, Osaka, Japan).
5
Transgenic Mouse Genotyping Protocol
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Traditional DI and piggyBac TS were analyzed by the same method. Expression of EGFP fluorescence in newborn transgenic mice was visualized by UV microscopy (VB-6000; KEYENCE Inc., Osaka, Japan) (Fig. 6 ). PCR genotyping of offspring was performed using a crude lysate of the tail tips (Fig. S4 ). Genomic DNA was extracted from the mouse tail using Direct PCR Lysis Reagent (Viagen Biotech Inc., Los Angeles, CA, US) containing 0.2 mg/ml proteinase K (Nacalai Tesque Inc., Kyoto Japan). The DNA lysate was used as a template in a 20-µl PCR reaction volume using go-Taq premixture (Promega Corporation) in the presence of the following primers to amplify the EGFP transgene and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. EGFP forward 5′-GCGACGTAAACGGCCACAAG-3′, EGFP reverse; 5′-TAGGTCAGGGTGGTCACGAG-3′. GAPDH forward 5′-ACCACAGTCCATGCCATCAC-3′, GAPDH reverse; 5′-TCCACCACCCTGTTGCTGTA-3′.
6
Comprehensive Histological and Immunohistochemical Analysis of Mouse Tissues
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Images of the mice and their organs were obtained using a digital fluorescent microscope (VB-6000; Keyence Japan, Osaka, Japan, or Leica AF6500; Leica Microsystems, Tokyo, Japan). For histological analyses, tissues were fixed in 10% formalin solution and embedded in paraffin for hematoxylin and eosin (H&E) staining. Alternatively, tissues were fixed by perfusion with 4% paraformaldehyde. Frozen sections were the cut using a Microm HM500 OM Microtome Cryostat (Carl Zeiss Japan, Tokyo, Japan).
The following primary antibodies were used for immunohistochemical analysis: guinea pig anti-insulin, rabbit anti-glucagon, rabbit anti-somatostatin, goat anti-pancreatic polypeptide, rabbit anti-chromograninA (cgA), rabbit anti-Ki67, and rabbit anti-VEGF (Abcam, Tokyo, Japan). Alexa568- or Cy3- labeled species-specific anti-IgG antibodies (Life Technologies Japan, Tokyo, Japan) were used as secondary antibodies. Images were obtained using a LSM710 laser scanning microscope (Carl Zeiss Japan), or a HS BZ-9000 fluorescent microscope system (Keyence). The Ki-67 index was calculated by dividing the total number of nuclei by the number of Ki-67-positive nuclei. The number of nuclei was counted by observing 8–10 fields with a 40× lens.
The following primary antibodies were used for immunohistochemical analysis: guinea pig anti-insulin, rabbit anti-glucagon, rabbit anti-somatostatin, goat anti-pancreatic polypeptide, rabbit anti-chromograninA (cgA), rabbit anti-Ki67, and rabbit anti-VEGF (Abcam, Tokyo, Japan). Alexa568- or Cy3- labeled species-specific anti-IgG antibodies (Life Technologies Japan, Tokyo, Japan) were used as secondary antibodies. Images were obtained using a LSM710 laser scanning microscope (Carl Zeiss Japan), or a HS BZ-9000 fluorescent microscope system (Keyence). The Ki-67 index was calculated by dividing the total number of nuclei by the number of Ki-67-positive nuclei. The number of nuclei was counted by observing 8–10 fields with a 40× lens.
7
Neuronal Apoptosis Quantification in Ischemic Brain
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The harvested brain was immersed in 4% paraformaldehyde for 2 days and embedded in paraffin. Four-micrometer-thick coronal sections at the level of the striatum (A 1.0-mm to P 1.0-mm of the bregma) were prepared for subsequent analyses. Deparaffinized sections were processed through antigen retrieval for 2 minutes in a pressure pot.
Fluorescence immunohistochemistry was performed (n=5/each group).
In order to assess treatment effects on neuronal apoptosis, sections were double labeled for CC3, the marker for apoptotic cells, plus NeuN, the marker for neuronal cells. Sections were reacted with primary antibodies against CC3 (rabbit monoclonal, Fluorescent signals were observed through appropriate filters using a fluorescence microscope (BX61; Olympus, Tokyo, Japan) and digitally photographed using a cooled charged-couple device camera (model VB-6000; Keyence Corporation, Osaka, Japan).
In the quantitative analysis, 2 and 4 ROIs were randomly set in the areas of the ischemic core and penumbra, respectively. The area of the penumbra was defined as the area where the ischemic region spared when a suitable treatment, i.e., transarterial reginal hypothermia in this study, was performed (Shichinohe et al., 2015; Takahashi et al., 2012) (Fig. 6B).
Fluorescence immunohistochemistry was performed (n=5/each group).
In order to assess treatment effects on neuronal apoptosis, sections were double labeled for CC3, the marker for apoptotic cells, plus NeuN, the marker for neuronal cells. Sections were reacted with primary antibodies against CC3 (rabbit monoclonal, Fluorescent signals were observed through appropriate filters using a fluorescence microscope (BX61; Olympus, Tokyo, Japan) and digitally photographed using a cooled charged-couple device camera (model VB-6000; Keyence Corporation, Osaka, Japan).
In the quantitative analysis, 2 and 4 ROIs were randomly set in the areas of the ischemic core and penumbra, respectively. The area of the penumbra was defined as the area where the ischemic region spared when a suitable treatment, i.e., transarterial reginal hypothermia in this study, was performed (Shichinohe et al., 2015; Takahashi et al., 2012) (Fig. 6B).
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