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.
Ab6720
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab6720 is a laboratory equipment product. It is designed for use in various research and scientific applications.
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Lab products found in correlation
Pk 4000, by Vector Laboratories (3 mentions)
Roti histol, by Carl Roth (2 mentions)
Dako retrieval solution, by Agilent Technologies (2 mentions)
Anti nrf2 sc 722, by Santa Cruz Biotechnology (2 mentions)
Ab6788, by Abcam (2 mentions)
Ab124267, by Abcam (2 mentions)
Immpact dab peroxidase substrate, by Vector Laboratories (2 mentions)
Nb810 56910, by Novus Biologicals (2 mentions)
Dm6000b microscope, by Leica (2 mentions)
Ab264429, by Abcam (1 mentions)
Ab64238, by Abcam (1 mentions)
Streptavidin horseradish peroxidase, by Abcam (1 mentions)
H 3403 500, by Vector Laboratories (1 mentions)
Ab206386, by Abcam (1 mentions)
Ab9533, by Abcam (1 mentions)
Ab6746, by Abcam (1 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
K346711, by Agilent Technologies (1 mentions)
Ab7403, by Abcam (1 mentions)
Ab215166, by Abcam (1 mentions)
27 protocols using ab6720
1
Quantifying Renal Cell Proliferation and Apoptosis
2
Intracellular HSV Protein Immunostaining
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Intracellular HSV protein was evaluated by immunostaining. Cells were fixed with 60/40 ice cold methanol/acetone for 15 min. Staining was performed using a rabbit polyclonal antibody directed against HSV-1 (ab9533) and a sheep polyclonal antibody directed against HSV-2 (ab21112) in combination with biotin-conjugated secondary goat anti-rabbit (ab6720) and rabbit anti-sheep (ab6746) antibodies (all antibodies derived from Abcam, Cambridge, United Kingdom). Protein was visualized using streptavidin peroxidase complex with AEC as a substrate.
3
Immunohistochemical Analysis of VAP-1 Expression
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Immunohistochemistry experiments (labeled streptavidin-biotin method) were performed using a Streptavidin-Peroxidase Immunohistochemical Detection kit (SP-9000; ZSGB-BIO, Beijing, China). In brief, formalin-fixed, paraffin-embedded sections (thickness: 4 µm) were dewaxed with fresh xylene and subjected to gradient hydration (100% ethanol, 95% ethanol, 60% ethanol, purified water). Following elimination of endogenous peroxidase activity (incubation with 3% H2O2 for 15 min at room temperature), microwave antigen retrieval and incubation with normal goat serum (Abcam) for 15 min at 37°C. The sections were then incubated overnight at 4°C with a primary antibody against VAP-1 (rabbit polyclonal; 1:200; ab187202; Abcam). Following 3 washes with PBS, the specimens were incubated with a biotin-conjugated goat anti-rabbit (1:1,000; ab6720; Abcam) secondary antibody for 15 min at 37°C, followed by streptavidin-peroxidase for 15 min at 37°C. The slides were stained with 3,3-diaminobenzidine for 10 min at room temperature, and re-stained with hematoxylin. The specimens were subsequently dehydrated in a graded series of ethanol (30, 70 and 100%), sealed with neutral resin, and observed under a light microscope (Olympus Corporation, Tokyo, Japan). A total of 10 fields were assessed (magnification, ×100) in three independent experiments.
4
Chondrocyte Proliferation and Hypertrophy Assay
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Proliferation and hypertrophy were detected with Ki67 and ColX immunohistochemistry staining respectively. Briefly, slides were dewaxed, dehydrated and hydrated before blocking cellular peroxidase activity with H2O2. For Ki67, an antigen retrieval step (10 m Sodium citrate pH6 + 0,05% Tween-20) was performed. Primary antibodies targeting Ki67 (Abcam ab16667) or ColX (Cosmo Bio LS-LB-0092) were incubated overnight in blocking buffer (TBS with 5% goat serum). The next day, the biotynilated secondary antibody (Abcam ab6720) was incubated for 30 min. An ABC kit (Vectastain PK4000) was added to amplify the biotin-streptavidin reaction and provide the detection by HRP and the DAB substrate (DAKO K346711). Slides were counterstained with Haematoxylin and mounted with Pertex.
5
Immunohistochemical Analysis of Immune Markers
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Mouse tumor samples were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue slices were deparaffinized, rehydrated, and antigen retrieval was performed using 10 mM citrate buffer (pH 6.0). CD80 (polyclonal rabbit, 1:200, ab215166, Abcam), CD86 (monoclonal rabbit, 1:200, ab243887, Abcam), and CD69 (polyclonal rabbit, 1:250, A00529-2, Boster) were stained overnight at 4 °C. The samples were washed and then incubated with goat anti-rabbit biotinylated secondary antibody (1:1000, ab6720, Abcam) and visualized using a horseradish peroxidase (HRP)-conjugated ABC system (1:10,000, ab7403, Abcam).
6
Immunohistochemical Analysis of Kidney Tissue
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Kidney sections (3 µm) (RM 2164, Leica, Wetzlar, Germany) were mounted on glass slides, heated at 60 °C for 1 h and deparaffinized with Roti-Histol (Roth, Karlsruhe, Germany) and ethanol. Antigen retrieval was performed with citrate buffer (DAKO Retrieval Solution, pH 6.0, Agilent Technologies, Santa Clara, CA, USA) at 95 °C for 30 min. Slides were then blocked and incubated overnight at 4 °C with the appropriate primary antibodies. The specific antibodies and dilutions were as follows: anti-γ-H2AX (#9718, 1:200, Cell Signaling, Herts, UK), anti-Nrf2 (sc-722, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-pNrf2 (S40, ab76026, 1:1000, abcam, Cambridge, UK). Sections were next incubated with the biotinylated secondary goat anti-rabbit antibody (ab6720, 1:200, abcam, Cambridge, UK) for 45 min at room temperature. Antibody binding was visualized as previously described [11 (link),29 (link)]. Sections were counterstained with hematoxylin. Images were acquired at 200-fold magnification. The ratio of positive to negative nuclei or areas was scored via ImageJ [30 (link)] within 10 visual fields of the cortex and 3–5 visual fields of the medulla.
7
Serum PTN and TNF-α Quantification
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The blood samples were collected via venipuncture into tubes containing anticoagulants. The protein level of PTN in serum was determined on covered 96-well ELISA plates, as previously reported (32 (link)). Rabbit anti-human PTN monoclonal antibodies (1:2,000, ab14025; Abcam, Cambridge, MA, USA) were diluted in Tris-buffered saline (TBS) and incubated at 4°C overnight. Biotinylated affinity-purified anti-rabbit secondary antibody (1:50,000, ab6720; Abcam) was added into the wells and incubated at room temperature for l h. Streptavidin/alkaline phosphatase conjugate (Roche Diagnostics GmbH, Mannheim, Germany) was added and maintained at room temperature. The absorbance at 405 nm was measured on a plate reader. Recombinant human PTN (R&D Systems, Inc.) was used as a standard control. The concentration of TNF-α was also measured using an ELISA kit (ab181421; Abcam) according to the protocols. The kit mainly consists of anti-TNF-α antibodies and the regent for the ELISA assay. The absorbance of TNF-α was determined at 450 nm.
8
Immunohistochemical Analysis of Testicular Apoptosis
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IHC was performed according to a previously reported method (Li et al., 2015 (link)). Briefly, testicular sections were dewaxed with xylene and dehydrated in serially graded ethanol solutions. The endogenous peroxidase activity was quenched with 3% H2O2 in distilled water. The sections were applied with anti-Bcl-2 antibody (ab196495, Abcam Inc., Cambridge, MA, USA; 1:50), anti-Cleaved caspase-3 antibody (AB3623, Merck Millipore, Billerica, MA, USA; 1:20), and anti-Cytochrome C (CytC) antibody (ab13575, Abcam Inc., Cambridge, MA, USA; 1:100) separately and then incubated overnight at 4°C. As a negative control, some sections were reacted with PBS instead of the specific antibodies. After reacting with a biotin-conjugated secondary antibody (ab6720, Abcam Inc., Cambridge, MA, USA; 1:1,000) for 1 h at room temperature, the sections were stained using the FAST DAB Peroxidase Substrate (Sigma, St Louis, MO, USA) and counterstained with haematoxylin for 10 s. The slides were then dehydrated and mounted for observation.
9
Immunohistochemical Analysis of Tumor Tissues
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The tumor tissues were fixed with 4% formaldehyde at 4 ˚C overnight and then embedded with paraffin. The tissues were sectioned into 4–5 µm slices. For hematoxylin and eosin (HE) staining, the dewaxed sections were stained with hematoxylin (Sigma, St. Louis, MO, USA) for 15 Min, and then incubated in hydrochloric acid alcohol (1%) for 15 Sec. After soaking in eosin (Sigma) for 2 Min, the slices were observed under an optical microscope. For theimmunohistochemical (IHC) assay, the sections were stained with F4/80 macrophage marker, EZH2, CCL5, or MMP2 (1:100, ab100790; Abcam, USA). In brief, after deparaffinized with xylene, the sections were subject to retrieval in a heating citrate buffer (pH 6.0) for 15 Min. After cooling down to temperature, the sections were blocked with normal goat serum (Thermofisher) at 37 ˚C for 10 Min and then incubated with primary anti‐F4/80, EZH2, CCL5, or MMP2 antibodies overnight at 4 °C. Next, sections were incubated with anti‐rabbit secondary antibody (biotinylated) (1:1,000, ab6720, Abcam) at room temperature for 30 Min. The horseradish peroxidase‐conjugated streptavidin (Amersham, Amersham, UK) was incubated with sections at 37 ˚C for 30 Min. The diaminobenzidine (Sigma) was used for chromogenic detection. The staining was observed under an inverted microscope (Olympus, Tokyo, Japan) (400× magnification).
10
Quantifying Liver Metastasis Angiogenesis
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Antibody staining was performed on histological sections of formalin-fixed liver metastases. Tumour sections were incubated overnight at 4 °C with the following primary antibodies: anti-VEGF (1:100, Abcam, ab1316, Shanghai, China) and anti-CD31 (1:100, Abcam, ab28364, Shanghai, China). The slides were rinsed and incubated in biotinylated secondary antibody (1:500, Abcam, ab6720 or ab6788, Shanghai, China). The intensity of staining for VEGF and CD34 expression was measured by Image-J with IHC profiler (Varghese et al. 2014 (link)).
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