TUNEL assays detecting apoptosis in tumor sections were performed with an ApopTag Plus Peroxidase in situ apoptosis kit (Chemicon) according to manufacturer’s instructions.
Apoptag plus peroxidase in situ apoptosis kit
Manufactured by Merck Group
Sourced in United States, Germany
The ApopTag Plus Peroxidase In Situ Apoptosis Kit is a laboratory product used to detect and quantify apoptosis, a form of programmed cell death, in tissue samples. The kit employs a terminal deoxynucleotidyl transferase (TdT) enzyme, which labels the fragmented DNA of apoptotic cells, allowing for their identification and analysis using peroxidase-based detection.
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64 protocols using apoptag plus peroxidase in situ apoptosis kit
1
Apoptosis Detection in Tumor Sections
2
Immunohistochemical Analysis of Pancreatic Tissues
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Pancreatic tissues were fixed in 10% (w/v) buffered formaldehyde, embedded in paraffin, cut at 4 μm thickness and applied on SuperFrost® slides (Menzel-Glaser, Braunschweig, Germany). Manual IHC was carried out as previously described [43 (link)]. The antibodies were used as followed: anti-ErbB2 (1:200, DAKO), anti-hCNT1 (1:100, H-70), and anti-MRP2 (1:100, sc5770, santa cruz). TUNEL assay was performed using ApopTag® Plus Peroxidase In Situ Apoptosis Kit (Chemicon) following manufacturer's protocol.
3
Detecting Apoptosis in 3D Epithelium
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Apoptotic cells in the epithelium were detected by ApopTag® Plus Peroxidase In Situ Apoptosis Kit (S7101, Chemicon®, Sigma-Aldrich, Tokyo, Japan) according to the manufacturer’s protocol. Briefly, the 3D epithelium equivalents were fixed by 10% formalin neutral buffer solution (062-01661, FUJIFILM Wako Chemicals, Osaka, Japan) at 4 °C overnight. The samples were then dehydrated by a series of ethanol and xylene followed by paraffin embedding. The paraffin tissue blocks were sectioned into 10 μm-thick slices by a microtome. After deparaffinization and hydration, apoptotic cells in the sample slices were detected by reagents of the TUNEL assay provided by the in situ apoptosis kit.
4
TUNEL Assay for Apoptosis Detection
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Apoptotic cells and bodies were detected by the terminaldeoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling (TUNEL) method (ApopTag® Plus Peroxidase In Situ Apoptosis Kit, Chemicon, Planegg-Muenchen, Germany). The TUNEL assay is regarded as the ‘gold standard’ in apoptosis detection and was performed as described previously [45 (link)-47 (link)].
5
Immunohistochemical Analysis of Tumor Markers
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Tumor sections were fixed and treated with 3% hydrogen peroxide for 30 min at room temperature. The tissues were incubated with anti-CD31 (PECAM-1, BD Biosciences), anti-mouse Ki67 (Clone MIB-5, Dako), 7A7 or 14F7 mAbs followed by LSAB®2 System, Peroxidase, DAB (Dako). The apoptosis was evaluated by ApopTag® Plus Peroxidase In Situ Apoptosis Kit (CHEMICON International). Negative controls were performed by substituting primary mAbs for the SSFT. Each staining included a known positive control: for murine EGFR: 3LL-D122 and X63 cells for NGcGM3. Finally, the sections were counterstained with Mayer’s hematoxylin.
6
Apoptosis and Tissue Necrosis Analysis
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The presence or absence of apoptotic cells within white syndrome tissue samples was confirmed using the in situ end labeling (ISEL) ApopTag Plus peroxidase in situ Apoptosis Kit (Chemicon International) as per the manufacturer's instructions. Adjacent tissue sections were also stained with Harris's haematoxylin and eosin (Sigma-Aldrich), Masson's Tri chrome (ProSciTech) and Alcian Blue/periodic acid-Schiff (ProSciTech) to determine tissue structures and evidence for necrosis of tissues. All tissue sections were permanently mounted following colour development.
7
TUNEL Assay for Apoptosis Detection
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TUNEL method was used in accordance with the user's manual of the manufacturer (Apoptag plus Peroxidase in situ Apoptosis Kit, Chemicon International, Cat: S7101). The procedure was following: Sections were incubated with proteinase K for 5 min, washed with distilled water and incubated with 3% hydrogen peroxide in PBS for 5 min. Later, sections were washed with PBS, put in the equilibrium buffer for 30 min and incubated in TdT enzyme at 37 °C for 1 hour. They were agitated in washing buffer for 15 s, washed in PBS, put into anti-digoxigenin conjugate for 30 min and then washed with PBS. After incubation with peroxidase for 6 min, they were washed with destilled water, stained with Mayer's haematoxylin and covered with mounting medium. In each section, TUNEL positive cells on 20 similar areas were counted under 20X magnifi cation.
8
Apoptosis Quantification in Ovarian Cancer
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The transfected SKOV-3 and OVCAR-3 cells were cultured for ~48 h at 37°C and 5% CO2. The apoptosis of the transfected cells was then detected by TUNEL assay as recommended in the ApopTag® Plus Peroxidase in Situ Apoptosis kit (Sigma-Aldrich; Merck KGaA). Briefly, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4°C. Subsequently, the cells were incubated with 1% TdT enzyme in a humidified atmosphere at 37°C for 90 min. Subsequently, the cells were stained with DAPI (Sigma-Aldrich; Merck KGaA) at 4°C for 10 min. Finally, the cells were observed under a fluorescence microscope (magnification, ×200). An average of 10 random fields with 100–200 nuclei per field was analyzed. The number of TUNEL-positive nuclei (green fluorescence) was expressed as the percentage of total nuclei (blue fluorescence).
9
Testicular Tissue Histology and Immunostaining
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For histological analyses, testes were fixed for 16 hours at 4°C in modified Davidson’s fixative (8.2% formalin, 33% ethanol, 11% glacial acetic acid in water). Fixed tissues were paraffin-embedded, sectioned at 5 μm, and then stained with the ApopTag Plus Peroxidase In Situ Apoptosis Kit (Sigma-Aldrich: S7101; St. Louis, MO, USA) or HE were used as previously described [18 (link)]. For immunohistochemical staining of testis sections or immunofluorescent staining of meiotic chromosome spreads, slides were blocked in PBS containing 0.15% Triton X-100, 10% BSA, 3% skim milk powder for 1 hour at RT, followed by incubation with primary antibodies for 16 hours at 4°C and detection by secondary antibodies (see antibody details in S1 Table ).
10
Tubular Epithelial Cell Apoptosis
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Apoptosis of tubular epithelial cells was detected with TUNEL staining with ApopTag Plus Peroxidase In Situ Apoptosis Kit (Sigma-Aldrich), according to the manufacturer’s instruction.
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