After being de-waxed and dehydrated, tissues in the 4 μm sections were eliminated endogenous peroxidase activity. Thereafter, sections were exposed to TUNEL reagent (TUNEL Assay Kit, ab206386, Abcam) for 1.5 h. Then, sections were dyed with DAB solution (ab206386, Abcam) for 10 min. Lastly, sections were observed under a microscope.
Ab206386
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab206386 is a rabbit polyclonal antibody specific for the target protein. This antibody is intended for use in immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Imagescope, by Leica (2 mentions)
Ab11174, by Abcam (1 mentions)
Aperio xt slide scanner, by Leica (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Ab264429, by Abcam (1 mentions)
Ab6720, by Abcam (1 mentions)
Ab64238, by Abcam (1 mentions)
Streptavidin horseradish peroxidase, by Abcam (1 mentions)
H 3403 500, by Vector Laboratories (1 mentions)
Bz x700, by Keyence (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
Neutral buffered formalin solution, by Fujifilm (1 mentions)
Methylbenzene, by Merck Group (1 mentions)
3 3 diaminobenzidine kit, by Solarbio (1 mentions)
Szx10, by Olympus (1 mentions)
Phox2b, by Abcam (1 mentions)
Autostainer plus, by Agilent Technologies (1 mentions)
Tunel assay kit, by Abcam (1 mentions)
Ihc tek epitope retrieval solution, by IHC World (1 mentions)
Envision detection systems peroxidase dab, by Agilent Technologies (1 mentions)
33 protocols using ab206386
1
TUNEL Assay for Apoptosis Detection
2
Immunohistochemical Analysis of Tissue Samples
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Four-micron sections of paraffin-embedded tissues were stained with Mayer's H&E Y/erythrosine B staining, Ki67 (0.084 μg/ml; 12202S; Cell Signaling Technologies, Danvers, MA, USA), and γH2A.X (0.06 μg/ml; ab11174; Abcam, Cambridge, UK) as described earlier (6 (link), 24 (link)) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis by immunohistochemical staining [ab206386; Abcam, Cambridge, UK) with Mayer's hematoxylin nuclear stain. Slides were imaged using an Aperio XT slide scanner and captured using Aperio ImageScope (Leica Biosystems, Wetzlar, Germany), and five high-power images were assessed per sample (N = 4–6 brains per treatment group] using ImmunoRatio (Seinajoki, Finland) as described earlier (24 (link)).
3
Quantifying Renal Cell Proliferation and Apoptosis
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Paraffin-embedded sections were used for Ki67 immunostaining (ab264429, Abcam, Cambridge, UK) to indicate cell proliferation. Briefly, sections were dewaxed in xylene and rehydrated by reducing concentrations of ethanol to distilled water. Antigen retrieval was performed in a microwave with sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched by incubating the slides with 3% hydrogen peroxide diluted in methanol, and washes were performed with tris-buffered saline supplemented with tween-20 (TBS-T). Blocking was performed with 10% goat serum, and antibody was applied overnight at 4 °C (1:500 in 5% goat serum diluted in TBS-T). The next day, slides were washed in TBS-T and incubated with goat anti-rabbit secondary antibody (1:1000, ab6720, Abcam) and streptavidin-horseradish peroxidase (1:500, S000-03, Rockland, PA, USA). Sections were stained with 3,3′-diaminobenzidine (ab64238, Abcam) and counterstained with nuclear fast red (H-3403-500, Vector). The level of cell apoptosis was determined using an HRP-DAB TUNEL assay kit (ab206386, Abcam) according to the manufacturer’s instructions. Ki67- and TUNEL-positive cells were determined in 100 randomly selected glomeruli and expressed as a ratio of the glomerular size.
4
TUNEL Staining for Apoptosis Detection
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TUNEL staining was performed with a TUNEL assay kit (ab206386, abcam). Briefly, tissues for the TUNEL stain were fixed in 4% neutral buffered paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. The slides were deparaffinized in lemosol and rehydrated by passage through graded alcohols. For antigen retrieval, the sections were incubated with Proteinase K solution. The sections were then incubated in 0.3% hydrogen peroxide in methanol for 20 min to inhibit endogenous peroxidase activity. Subsequently, the samples were labeled with TdT enzyme and FITC. After 3 washes with TBS-T, the sections were blocked with normal serum and incubated with anti-FITC antibody conjugated with HRP. After 3 washes with TBS-T, the signals were visualized with DAB chromogen solution. The slides were counterstained with hematoxylin. Histopathological images were acquired using a microscope (BZ-X700, KEYENCE).
5
TUNEL Staining in Paraffin Sections
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Paraffin-embedded tissues were deparaffinized in xylene and then rehydrated in graded ethanol. TUNEL staining was completed per manufacturer’s instructions (Abcam, Cat # ab206386). Briefly, slides were permeabilized with a proteinase K solution, and covered with TdT equilibration buffer. Tissues were then labeled with a TdT labeling reaction mixture for 1.5 h, and then a conjugate solution for 30 min. DAB solution was added for 15 min, and then slides were washed with water. Slides were counterstained with methyl green and mounted with permount. Images were obtained on a Leica DM6B with a ×20 objective.
6
Apoptosis Detection in MPTP-Induced PD
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TUNEL assay was performed to verify apoptosis that might appear in Purkinje cells of cerebellar tissues of MPTP-induced PD mice. A TUNEL kit (ab206386, Abcam, Cambridge, USA) commercially available was used for this analysis. All processes were performed according to the manufacturer’s directions. An optical microscope was used to take photomicrographs.
7
In Situ Apoptosis Analysis in AFL-Treated Skin
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AFL-treated skin was dissected, fixed in formalin, and subjected to paraffin embedding and sectioning. Paraffin sections were subjected to side-by-side H&E staining and in situ apoptosis analysis via a commercial kit (ab206386, Abcam). In brief, paraffin sections were deparaffinized in xylene and rehydrated in a graded alcohol series. After being treated with proteinase K, the sections were incubated with 3% H2O2 to inactivate endogenous peroxidases. Biotin labeling of exposed 3′-OH ends of DNA of apoptotic cells was generated by adding terminal deoxynucleotidyl transferase (TdT) enzyme and TdT labeling reaction mix (included in the kit) for 90 minutes at room temperature, and the reaction was stopped using the stop buffer. Biotinylated DNA was detected by incubation with a streptavidin-HRP conjugate (included in the kit) for 30 minutes at room temperature. After washing in TBS, DAB substrate was added, and reactions were stopped. The sections were counterstained with Methyl Green, dehydrated, and then coverslipped. Images were taken under a Nikon Eclipse E600 microscope.
8
Histological Assessment of Acute Pancreatitis
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Pancreatic tissues were fixed in a 10% (v/v) neutral buffered formalin solution (Wako, Osaka, Japan) for 24 h and embedded in paraffin. Paraffin sections were cut and stained with hematoxylin/eosin (H&E). The severity of AP was evaluated by two experienced pathologists blinded to the study protocol. The pancreatic structure, inflammation, and necrosis were semiquantitatively analyzed according to a scoring system described previously (Supplementary Table 1 ). Apoptotic cells were analyzed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining according to the manufacturer’s instructions (Abcam, ab206386). For a positive control, sections were treated with 1 µg/µl DNase I in TBS/1 mM MgSO4 for 20 min at room temperature. The specimens were examined by light microscopy (Olympus, BX53).
9
TUNEL Assay for Apoptosis Analysis
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Terminal transferase-mediated biotin 2′-deoxyuridine, 5′-triphosphate nick end labeling (TUNEL) assay was performed to evaluate apoptosis of cells with a TUNEL assay kit (ab206386, Abcam, Cambridge, UK) as previously described (27 (link)). The hippocampus tissue was first deparaffinized and treated with 20 μg/ml deoxyribonuclease-free proteinase K (AM2548, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 20 min and was then washed with PBS three times (5 min for each). After being fixed at room temperature for 15 min, the samples were washed with PBS for another three times (5 min each time). Next, each sample was added with 50 μl of biotin labeling and incubated for 1 h at 37°C, followed by processing with a 3,3′-diaminobenzidine kit (DA1015, Solarbio, Beijing, China) and PBS washing. Then, the samples were sealed after clearing with methylbenzene (244511, Sigma-Aldrich, USA) for 5 min. The apoptotic cells were observed using a stereomicroscope (SZX10, Olympus, Japan) under × 100 and × 200 magnifications.
10
Mouse Xenograft Model for GC Therapy
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All animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University (Approval number: 2019-058). An experimental mouse xenograft model was established by subcutaneous injection of 5 × 106 GC cells with or without PSMD7 knockdown (n = 5 per group) in 100 μL of PBS into 6-week-old female nude mice. Tumor volumes were recorded every 4 days and calculated according to the following formula: volume = (width2 × length)/2. One week after implantation, the mice were treated with DDP (5 mg/kg) in PBS (thrice a week) via intraperitoneal injection. Four weeks after implantation, the mice were sacrificed, and xenograft tumors were collected for IHC and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (ab206386, Abcam) following the manufacturer's instructions.
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