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6 protocols using m7248

1

Quantifying Arterial Plaque Composition

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Carotid cross sections were stained for SR (Direct Red 80, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany; 365548), ki67 (with the antibody M7248 from Dako, Agilent Technologies, Santa Clara, CA, USA; 1:10 diluted) and SMA (M0851; Dako; 1:100 diluted). These sections were scanned. Subsequent analysis was conducted with QuPath software (version 0.2.3, Queen’s University, Belfast, UK) [61 (link)]: once more, the areas of media and neointima were first designated manually before the subsequent parameters were automatically quantified for these two areas. The positive cell detection command in QuPath was used for cell detection, counting and classification. In the ki67-stained sections, the overall cell number, as well as the cell density per mm² and the number of ki67+ cells per cross-section were determined. The threshold for positive cell detection was set using the pixel intensity histogram in order to ensure a standardized classification while still taking staining variation between slides into account. The formula used was:
The relative proportion of SMA+ and SR+ areas was measured. For quantification of SMA-positive cells, it is common to analyze the stained areas [62 (link),63 (link)], as a reliable assignment to individual cells is not possible.
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2

Tissue Preparation and Immunofluorescent Staining

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Paraffin-embedded tumor issues were sliced into 5-mm thick section and tissue slides were deparaffinized in Tissue-Clear (1466; Sakura) and rehydrated with sequential immerse in 99–95–70% ethanol. Frozen tissue samples embedded in the OCT Embedding Compound (SAKURA) were sectioned using a cryostat, followed by fixation with 4% PFA for 20 min. Tissue slides were stained with a rat anti-mouse endomucin (1:400; 14-5851-85; eBioscience) antibody, a mouse anti-human αSMA (1:200; M0851; clone 1A4; DAKO) antibody, a rabbit anti-mouse NG2 (1:400; AB5320; Millipore) antibody, and a rat anti-mouse Ki67 (1:100; M7248; DAKO) antibody, followed by staining with species-matched secondary antibodies as follows: an Alexa Fluor 555-labeled goat anti-rat (1:400; A21434; Invitrogen), an Alexa Fluor 488-labeled donkey anti-mouse (1:400; A21202; Invitrogen), and an Alexa Fluor 488-labeled donkey anti-rabbit (1:400; A21206, Invitrogen). Positive signals were detected using a fluorescence microscope equipped with a camera (Nikon, DS-QilMC). Images were analyzed using an Adobe Photoshop software (CS5; Adobe) program. Vascular coverage was quantified as a percentage of vessels covered by αSMA+ cells by calculating overlapping area of endomucin and αSMA+ positive signals.
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3

Quantifying BRCA1, CTSS, and Ki-67 in Breast Cancer

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Immunohistochemistry was performed with the following antibodies: BRCA1 (ab16780, Abcam) or CTSS (sc-271619, Santa Cruz Biotechnology) and mouse anti-Ki-67 (M7248, Dako). Human breast cancer tissue slides were purchased from US Biomax. For antigen retrieval, slides were placed in citric acid buffer (pH 6.0) and heated at 100 °C for 10 min. Slides were incubated overnight at 4 °C with antibodies. Quantification of images was measured with image analyzer (Image J, NIH, Bethesda, MD, USA). All statistical analyses of images were performed using GraphPad Prism software 5.0 (GraphPad Software, San Diego, CA, USA).
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4

Immunohistochemistry of Paraffin Sections

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Paraffin sections with thicknesses of 3 µm were processed for immunohistochemistry. For labeling with primary antibodies, the sections were deparaffinized and treated for 30 min with 0.3% H2O2 in methanol. All the sections were heated in an autoclave for 20 min in a citrate buffer (pH 6.0), then incubated with the primary antibodies overnight. The primary antibodies and conditions are as follow: anti-p21 (1:25, mouse monoclonal, NBP2-29463, Novus Biologicals, CO, USA); anti-Ki-67 (1:800, mouse monoclonal, M7248, Dako, CA, USA), anti-CD31 (1:60, goat monoclonal, AF3628, R&D Systems, MN, USA), and anti-desmin (1:80, mouse monoclonal, M0760, Dako, CA, USA). Sections were stained with biotinylated anti-mouse IgG or anti-goat IgG (Dako, CA, USA) and visualized using H2O2-containing diaminobenzidine buffer.
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5

Immunohistochemistry for Ki67 in Renal Tissue

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Tissue sections were deparaffinized in xylene and rehydrated in graded alcohol. Immunostaining for Ki67 was carried out by using a DAKO LSAB2 kit (K0609) according to the manufacturer's instructions. Briefly, tissue sections were heated at 121°C for 15 min using an autoclave, and citrate buffer (pH 6.0) was used for antigen retrieval. The sections were treated for 30 min with methanol to which 1% H2O2 was added to inactivate endogenous peroxidase activity. The sections were then incubated for 30 min at room temperature with anti‐Ki67 (M7248; DAKO) diluted 1:50 by DAKO Antibody Diluent (S2022; DAKO). After washing, slides were incubated with biotinylated anti‐mouse immunoglobulins and horseradish‐conjugated streptavidin (LSAB2, K0609; DAKO), each for 10 min. Then, DAB substrate (K3466; DAKO) was applied to develop the stain. Sections were washed and counterstained with hematoxylin. Ki67‐positive cells were counted in 50 randomly selected glomeruli and in the tubular epithelium of the renal cortex in 20 microscope fields per section at ×400 magnification as described in a previous paper (Johnson et al. 2006).
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6

Multicolor Immunohistochemistry for Glioma

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Vibratome sections (200 μm thick) of mouse brains were paraffin-embedded and then consecutive semithin sections were processed and incubated with primary antibodies against Vimentin, glial fibrillary acidic protein (GFAP) and Ki67 (M7248, DAKO) followed by horseradish peroxidase-conjugated secondary antibodies (K4065, K5007, DAKO) and visualized by a chromogen or stained with hematoxilin-eosin. Slides containing tumor tissue from GBM patients or neurospheres from GSC cultures embedded in paraffin were stained with antibodies against the N-terminal region of ODZ1.
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