Tumor tissues were embedded in paraffin, sectioned at 4 μm, then cell proliferation ability was assessed by quantification of Ki-67 staining (percentage of positive cells). In brief, antihuman Ki-67 antibody (BD Pharmingen, CAT 550609) and p-ERK (Cell Signaling Technology, CAT#4370) were 1:200–300 diluted, and immunostaining was done according to a standard protocol using DAB Substrate Kit (ZSGB-BIO). To ensure the comparability of immunohistochemical staining, a common reference standard was included as an internal or intra-assay control in each batch. Ki-67 protein expression was scored with 0, 1, 2, 3, which represent negative, weak positive, positive, and strong positive, respectively. Ki-67 protein expression scored with 1, 2, 3 were calculated as positive, the percent of positive staining cells were then calculated. ERK phosphorylation was quantitated by integral optical density (IOD) using Image-pro plus 6.0 (Media Cybernetics). Each stained section was evaluated under the same magnification, light brightness, and exposure intensity. To evaluate the effect of different treatments on different organs, we performed hematoxylin and eosin (H&E) staining of liver, kidney, and heart sections.
Antihuman ki 67 antibody
Manufactured by BD
The Antihuman Ki-67 antibody is a laboratory reagent used for the detection of the Ki-67 protein, which is a marker of cellular proliferation. The antibody is designed for use in immunohistochemical and flow cytometry applications.
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3 protocols using antihuman ki 67 antibody
1
Quantitative Analysis of Cell Proliferation
2
Quantitative Assessment of Cell Proliferation
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Tumor tissues were embedded in paraffin, sectioned at 4 μm, then cell proliferation ability was assessed by quantification of Ki-67 staining (percentage of positive cells). In brief, antihuman Ki-67 antibody (BD Pharmingen, CAT 550609) and p-ERK (Cell Signaling Technology, CAT#4370) were 1:300 diluted, and immunostaining was done according to a standard protocol using DAB Substrate Kit (ZSGB-BIO). To ensure the comparability of immunohistochemical staining, a common reference standard was included as an internal or intra-assay control in each batch. Ki-67 protein expression was scored with 0, 1, 2, and 3, which represent negative, weak positive, positive, and strong positive, respectively. Ki-67 protein expression scored with 1, 2, and 3 were calculated as positive, the percent of positive staining cells were then calculated. ERK phosphorylation was quantitated by integral optical density (IOD) using Image-pro plus 6.0 (Media Cybernetics, USA). Each stained section was evaluated under the same magnification, light brightness, and exposure intensity. To evaluate the effect of different treatments on different organs, we performed hematoxylin and eosin (H&E) staining of liver, kidney, and heart sections.
3
Xenograft Tumor Growth Inhibition
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Female athymic nude mice were purchased from SLAC laboratory Animal Co., Ltd.
(Shanghai, PR. China) and housed in a specific pathogen-free (SPF) environment.
C643 cells (3×10 6 ) were injected subcutaneously into the flanks of mice at the age of 5 weeks. When tumors grew to 5 mm in diameter, mice were divided into two groups (five mice per group) and administered the following treatments: vehicle control (DMSO) or KA, 1 mg/kg through intraperitoneal injection daily for five days.
Tumor volume was measured every 2 days during the course of the therapy, and was calculated by the formula (width) 2 × length/2. After treatment for 7 days, tumors were harvested and weighted. Tumor tissues were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E). Cell proliferation ability was assessed by quantification of Ki-67 staining (percentage of positive cells).
In brief, anti-human Ki-67 antibody (BD Pharmingen) was 1:150 diluted and immunostaining was done according to a standard protocol using DAB Substrate Kit (ZSGB-BIO). At least 1000 Ki-67-positive cells were scored by visual examination of 5 randomly selected fields (×200 magnification). In addition, to evaluate the effect of different treatments on animal hepatocytes and kidney, we performed H&E staining of liver and kidney sections.
(Shanghai, PR. China) and housed in a specific pathogen-free (SPF) environment.
C643 cells (3×10 6 ) were injected subcutaneously into the flanks of mice at the age of 5 weeks. When tumors grew to 5 mm in diameter, mice were divided into two groups (five mice per group) and administered the following treatments: vehicle control (DMSO) or KA, 1 mg/kg through intraperitoneal injection daily for five days.
Tumor volume was measured every 2 days during the course of the therapy, and was calculated by the formula (width) 2 × length/2. After treatment for 7 days, tumors were harvested and weighted. Tumor tissues were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E). Cell proliferation ability was assessed by quantification of Ki-67 staining (percentage of positive cells).
In brief, anti-human Ki-67 antibody (BD Pharmingen) was 1:150 diluted and immunostaining was done according to a standard protocol using DAB Substrate Kit (ZSGB-BIO). At least 1000 Ki-67-positive cells were scored by visual examination of 5 randomly selected fields (×200 magnification). In addition, to evaluate the effect of different treatments on animal hepatocytes and kidney, we performed H&E staining of liver and kidney sections.
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