TUNEL staining was conducted as described46 (link). Deparaffinized sections were incubated with proteinase K and DNA fragments were labeled with fluorescein-conjugated dTUP using in situ Apoptosis Detection kit (MK500, Takara). Nuclear density was determined by manual counting of DAPI-stained nuclei in 10 fields for each animal using a 40x objective.
Mk500
Manufactured by Takara Bio
Sourced in Japan
The MK500 is a versatile laboratory instrument designed for automated DNA and RNA extraction. It utilizes magnetic bead-based technology to efficiently purify nucleic acids from a variety of sample types. The MK500 offers consistent and reliable performance, ensuring high-quality results for downstream applications.
Automatically generated - may contain errors
10 protocols using mk500
1
Apoptosis Detection by TUNEL Assay
2
TUNEL Staining of Apoptotic Cells
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We used in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit (MK 500, Takara, Shiga, Japan) to visualize apoptotic cells in the brain, heart, lungs, liver and kidney. Five micrometer thickness frozen sections were dried at room temperature for 10 min and rinsed 3 times in PBS for 5 min. Sections were incubated for 10 min with permeabilisation buffer. Sections were then incubated with the labeling reaction mixture (TdT enzyme 5 μl + labeling safe buffer 45 μl) for 90 min at 37°C in a humidified chamber. Slides were washed 3 times for 5 min in PBS to stop the reaction. TUNEL images were acquired using a fluorescence microscope (BX-53, Olympus, Tokyo, Japan). Positive cell counts per field were averaged across 20 fields per sample.
3
Apoptosis Evaluation in Retina Tissues
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TUNEL staining of 10 μm-thick retina tissues was conducted to evaluate apoptosis as described previously 44 (link). After washing with PBS, the tissues were blocked in 5% BSA plus 0.3% Triton X-100 for 1 h at room temperature, and treated with in situ detection kit according to the manufacturer's protocol (MK500, TaKaRa Bio Inc, Shiga, Japan) at room temperature for 1.5 h. The sections were then observed under Olympus SLIDEVIEWTM VS200 microscope (Olympus, Germany) and processed by Image J or Adobe Photoshop CC 14.0 software.
4
Histological and Cellular Analyses of Metabolic Tissues
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Fixed tissues were embedded in paraffin and sectioned. Kidney sections were stained with PAS, and liver and white adipose tissue sections were stained with hematoxylin and eosin. Pancreatic sections were stained with antibodies against insulin (1:500, Abcam, #ab7842) and glucagon (1:100, Sigma-Aldrich, #G2654). TUNEL staining was performed in accordance with the manufacturer's instructions (TaKaRa, #MK500). Immunofluorescent staining of the hypothalamus was performed with an anti-phosphorylated AMPKThr172 antibody (1:50, Cell Signaling Technology, #2531). The rat ventromedial hypothalamic nucleus (VMH) was sectioned 2.56 mm posterior to the bregma, ± 0.4 mm laterally and 9.5 mm from the dural surface (Satoh et al., 1997 (link)). Podocyte structure was evaluated by scanning electron microscopy (Hitachi S-570, Tokyo, Japan). To analyze the β cell area in pancreatic sections, the proportion of the insulin-positive area in the total pancreatic area was evaluated in each insulin-stained section using ImageJ software. Seven pancreatic sections were analyzed in each animal. The apoptotic index was calculated as a percentage of TUNEL-positive cells using the following formula: apoptotic index = 100 × (number of TUNEL-positive cells / number of insulin-positive cells).
5
Histological Analysis of Tumor Apoptosis
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Tumor tissues were fixed in 4% formalin, embedded in paraffin, cut into 4 μm slices, and stained with hematoxylin and eosin (H&E) or indicated antibodies. Apoptotic cells in tumor sections were analyzed by an in situ Apoptosis Detection Kit (TUNEL, Takara, MK500). Color changes of the slides were photographed using light microscope with companion software (Leica, Germany).
6
Quantifying Apoptotic Cells in Cerebral Cortex
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Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive nuclei were detected by using an in situ Apoptosis Detection Kit (MK500; Takara Bio Inc., Shiga, Japan). Surviving cells and TUNEL-positive nuclei in the cerebral cortex were counted under ×400 magnification (IX-71; Olympus) in 5 sections per animal, which corresponded to coronal coordinates of −2.3 to 0.70 from bregma. Images (657.34 × 863.41 µm) were assessed by quantifying the number of stained cells as a percentage of total number of cells with the Multi Wavelength Cell Scoring Module of MetaMorph software (Molecular Devices).
7
Histological and Immunohistochemical Analysis of Cultured Vessels
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Cultured blood vessels and spheroids were fixed in 4% PFA (immunohistochemistry) or Bouin's fixative (HE staining) overnight. Fixed specimens were dehydrated using 70% ethanol in situ, detached from the culture dish, and transferred to a glass bottle. The samples were further dehydrated in graded concentrations of ethanol. Then, the ethanol was substituted with xylene and paraffin. The paraffin block was cut at 10 μm thicknesses. For histology, these sections were stained with hematoxylin and eosin.
For immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), sections were deparaffinized and stained using the protocols provided by the manufacturers. Antibodies against PDGFB (Abcam, ab23914), type IV collagen (LSL, LB-0445), and desmin (LabVision, MS-376-S0) were used. To detect apoptotic cells, we used an In situ Apoptosis Detection Kit (Takara Bio, MK500).
For immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), sections were deparaffinized and stained using the protocols provided by the manufacturers. Antibodies against PDGFB (Abcam, ab23914), type IV collagen (LSL, LB-0445), and desmin (LabVision, MS-376-S0) were used. To detect apoptotic cells, we used an In situ Apoptosis Detection Kit (Takara Bio, MK500).
8
Histopathological Evaluation of i-PDT Effects
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Tissue sections were subjected to hematoxylin eosin (HE) staining for morphological analysis of the tissue. For the evaluation of i-PDT-induced tissue changes, in addition to the morphological changes in the tumor tissue, the area of tissue changes that may be a result of i-PDT was measured using NDP.view2 software (Hamamatsu Photonics K.K., Hamamatsu, Shizuoka, Japan). For evaluation of the tumor-killing effects of i-PDT, the TUNEL method (in situ apoptosis detection kit MK500, Takara Bio, Japan) was used in which the label was a rabbit-derived anti-fluorescein isothiocyanate (FITC) horseradish peroxidase (HRP) conjugate and 10 × diluted terminal nucleotidyl transferase, and the chromogenic substrate was a 50 × diluted 3,3'-diaminobenzidine (DAB) solution. DAB staining was performed for nuclear staining. The effect of i-PDT on tumor blood vessels was analyzed by phosphotungstic acid hematoxylin (PTAH) staining for the presence of intravascular thrombi and vascular endothelial injury was analyzed by immunohistochemical staining with anti-CD31 antibody (Dianova, Biozol Co., Hamburg, Germany).
9
Retinal Apoptosis Evaluation by TUNEL
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Retinas were frozen, sectioned, and subjected to the TUNEL assay using an in situ apoptosis detection kit (MK500, Takara Bio, Shiga, Japan).
10
In Situ Apoptosis Detection
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Apoptosis was evaluated using an in situ apoptosis detection kit (mk500, Takara, Japan). Proteolysis was performed using proteinase K for 15 min. Working Strength TdT enzyme was added to the proteolyzed slices (5 μL TdT enzyme: 45 μL reaction buffer). Then, the slices were incubated in a humidified chamber at 37°C for 90 min. Next, the slices were treated with fluorescein isothiocyanate‐antibody (FITC‐Ab) mixture in a humidified chamber at 37°C for 60 min. Finally, sections counterstained with Hoechst were observed using a digital fluorescence slide scanner. The areas with fluorescence were determined using the ImageJ 1.50e software (Bethesda).
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