Liver metastatic tissues were sliced at 10 μm thickness with a microtome and stained overnight at 4 °C with an anti-Ki67 antibody (Novus Biologicals, Littleton, CO, USA) and EG-VEGF/PK1 antibody (Novus Biologicals, Littleton, CO, USA). For each positive cell counting, one field magnified 400-fold in each of the five vascularized areas was counted, and average counts were recorded. Since we confirmed that the outcome data were not normally distributed with the Kolmogorov–Smirnov test, the Mann–Whitney U test was used to determine any significant difference, which was set at p < 0.05. All statistical analyses were performed using EZR software 1.63 [18 (link)].
Anti ki67 antibody
Manufactured by Novus Biologicals
Sourced in United States
The Anti-Ki67 antibody is a laboratory reagent used for the detection and quantification of the Ki67 protein, a well-established marker of cellular proliferation. This antibody can be used in various immunohistochemical and immunocytochemical applications to assess the proliferative state of cells.
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Lab products found in correlation
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Evos fl auto imaging system, by Thermo Fisher Scientific (2 mentions)
Cytotoxicity assay kit, by Promega (2 mentions)
Nb500 170, by Novus Biologicals (2 mentions)
Ab210702, by Abcam (2 mentions)
Ix81 fluorescent microscope, by Olympus (1 mentions)
Anti pstat3 antibody, by Cell Signaling Technology (1 mentions)
Ultracut microtome, by Leica (1 mentions)
Anti socs3, by Cell Signaling Technology (1 mentions)
Axioskop2 mot plus microscope, by Zeiss (1 mentions)
Alexa fluor 594 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dab kit, by Merck Group (1 mentions)
Anti lysozyme, by Abcam (1 mentions)
Anti lgr5 antibody, by Abcam (1 mentions)
Anti villi, by Abcam (1 mentions)
Alexa fluor 594 conjugated goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd31 monoclonal antibody, by BD (1 mentions)
Real target retrieval solution, by Agilent Technologies (1 mentions)
Tcs sc8, by Leica (1 mentions)
12 protocols using anti ki67 antibody
1
Quantifying Liver Metastasis Proliferation
2
Multimodal Analysis of Intestinal Tumor Samples
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HE staining, IHC, and IF were performed as described previously [19 (link), 20 (link)]. IHC employed the following reagents: anti-Ki67 antibody (Novus Cat. NBP1-40684), anti-SOCS3, and anti-p-Stat3 antibodies (Cell Signaling Technology). IF employed the following reagent: anti-p-Stat3 antibody (Cell Signaling Technology).
For the RNA-FISH assay, the colonic tumor tissues from Apc+/min mice were recovered at necropsy, washed with PBS, fixed in 4% PFA buffer (paraformaldehyde diluted in PBS that had been pre-treated with Diethylpyrocarbonate). The fixed tissues then were embedded in paraffin. After sectioning with an Ultracut microtome (Leica, Bannockburn, IL, USA), the embedded tissues were dehydrated and digested with Protein K, pre-hybridized for 1 h, and hybridized with miR-708 probe overnight at 37 °C. The miR-708 probe consisted of a 6-carboxyfluorescein (FAM)-labeled oligonucleotide with the sequence 5′-FAM-GGGUCGAUCUAACAUUCGAGGAA-FAM-3′; the oligo was purchased from Guangzhou Ribobio Co., Ltd. (Guangzhou, China). After incubation with 4′,6-diamidino-2-phenylindole(DAPI) staining buffer for 10 min, the sections were mounted and then examined with an Olympus IX81 fluorescent microscope.
For the RNA-FISH assay, the colonic tumor tissues from Apc+/min mice were recovered at necropsy, washed with PBS, fixed in 4% PFA buffer (paraformaldehyde diluted in PBS that had been pre-treated with Diethylpyrocarbonate). The fixed tissues then were embedded in paraffin. After sectioning with an Ultracut microtome (Leica, Bannockburn, IL, USA), the embedded tissues were dehydrated and digested with Protein K, pre-hybridized for 1 h, and hybridized with miR-708 probe overnight at 37 °C. The miR-708 probe consisted of a 6-carboxyfluorescein (FAM)-labeled oligonucleotide with the sequence 5′-FAM-GGGUCGAUCUAACAUUCGAGGAA-FAM-3′; the oligo was purchased from Guangzhou Ribobio Co., Ltd. (Guangzhou, China). After incubation with 4′,6-diamidino-2-phenylindole(DAPI) staining buffer for 10 min, the sections were mounted and then examined with an Olympus IX81 fluorescent microscope.
3
Evaluating eNano-Ro5 Cytotoxicity and Proliferation on Human Retinal Endothelial Cells
4
Methotrexate Inhibits Proliferation of C-PVR Cells
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C-PVR cells were cultured on cover slips at a confluency of 30,000 cells per well and treated with different concentrations of MTX (100, 200, and 400 μM) or vehicle. At the 24-, 48-, and 72-hour time points, cells were washed and fixed with 4% PFA for 10 minutes. Cells were permeabilized with 0.5% Triton X-100 in PBS for 5 minutes and blocked (10% goat serum in PBS) for 1 hour at room temperature. Proliferation was evaluated by incubating cells with anti-Ki67 antibody (1:50; Novus Biologicals, Cambridge, United Kingdom). The primary antibody was prepared in antibody dilution buffer (5% goat serum) and incubated overnight at 4°C. Cells were incubated with goat anti-rabbit Alexa Fluor 594 secondary antibody (Life Technologies) at 1:300 dilution for 2 hours at room temperature. The cover slips were washed and mounted onto slides using Prolong Gold Antifade Reagent with DAPI. Images were obtained using a Zeiss Axioskop 2 MOT Plus microscope (Carl Zeiss, Inc.). The quantification is reported as an average of Ki67-positive cells to the number of DAPI-positive cells per high-powered field of human C-PVR cultures, which were cultured in triplicate.
5
Evaluating Cytotoxicity and Proliferation of Human Retinal Endothelial Cells Exposed to eNano-Ro5
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Human retinal endothelial cells were cultured to reach 90% confluency per well and were treated with different concentrations of eNano-Ro5 mixed with HREC media (0.5%, 1%, and 2%) or nanoemulsion without Ro5-3335 (vehicle) as a control. After 24 hours, supernatant was obtained and lactate dehydrogenase levels were measured to analyze cytotoxicity using the Cytotoxicity Assay kit (Promega). Proliferation was evaluated 48 hours after the treatment media was added. Cells were washed and fixed (4% paraformaldehyde for 10 minutes) before permeabilization (0.5% Triton X-100 in PBS for 5 minutes) and blocking (10% goat serum in PBS for 1 hour at room temperature). To assess proliferation, cells were incubated with anti-Ki67 antibody (1:50; Novus Biologicals) in antibody dilution buffer (5% goat serum) and incubated overnight at 4°C. Cells were incubated with secondary antibody goat antirabbit Alexa Fluor 594 (Life Technologies) at 1:300 dilution for 2 hours at room temperature. Cells were washed in PBS and stained using Prolong Gold Antifade Reagent with 4',6-diamidino-2-phenylindole (DAPI). Images were obtained using an EVOS FL Auto imaging system (Life Technologies). The quantification is reported as an average of Ki67-positive cells to the number of DAPI-positive cells per high-powered field of HREC culture, which were cultured in triplicate.
6
Evaluating Proliferation of Y-27632-Treated pTM Cells
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The Ki67 marker was used to evaluate the proliferation of pTM cells exposed to Y-27632. PTM cells were seeded on coverslips in 24-well plates (1 × 103 cells/well) and incubated with or without 100 μM Y-27632 for 24 or 48 h. Cells were fixed in 4% formaldehyde for 15 min at RT, washed with DPBS, and permeabilized with 0.3% Triton X-100 in DPBS. Cells were then incubated in the blocking solution for 1 h. Samples were stained with rabbit polyclonal anti-Ki-67 antibody (1:200; Novusbio) at 4°C overnight, washed with DPBS, and incubated with Alexa FluorTM 568 goat anti-rabbit IgG (1:200) for 1 h at RT. Cells were counterstained with DAPI to stain the nuclei. The stained cells were then photographed using a confocal microscope (Nikon; Tokyo, Japan).
7
Immunohistochemical Analysis of Intestinal Stem Cells
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The 4‐μm‐thick sections of paraffin‐embedded small intestine sections were dewaxed and rehydrated with citrate buffer. Then, the sections were boiled in 10 mM/L citrate buffer solution (pH 9.0) for antigen retrieval according to standard procedures. After antigen retrieval, the sections were incubated with serum for 1 hour at room temperature to block non‐specific antigen‐binding sites; then, the sections were incubated with anti‐Lgr5 antibody (1:50 dilution; Abcam, Cambridge, MA), anti‐Ki67 antibody (1:300 dilution; Novus, Littleton, CO), anti‐lysozyme (1:800 dilution; Abcam) or anti‐villi (1:800 dilution; Abcam) overnight at 4°C. Sections were then incubated in secondary antibody for 30 minutes at 37°C. Positive cells were detected using a DAB kit (Sigma Aldrich, St. Louis, MO). The images were captured, and positive staining was quantified objectively by the integrated performance primitives (IPP) software as described previously in a blinded fashion.
8
Immunohistochemical Analysis of Malignant Mesothelioma
9
Immunohistochemical Analysis of Tumour Vasculature
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MIAPaCa-2 tumour tissues were surgically removed and immediately embedded and frozen in OCT compound. Cryostat sections (8 μm thick) were fixed in 4% paraformaldehyde for 10 min, blocked with 1% BSA in DPBS and then incubated with anti-Ki67 antibody (1:200 dilution, Novus Biologicals, Littleton, CO, USA) or rat anti-mouse monoclonal CD31 antibody (1:100 dilution, BD Biosciences, 550274). After extensive washing with DPBS, the sections were incubated with Alexa Fluor 594-conjugated goat anti-rat IgG (1:300 dilution, Invitrogen) for 1 h, counterstained with 1 µg/ml DAPI and observed under a confocal laser scanning microscope (Fluoview, Olympus). To determine the vessel density, pixel values of the CD31-positive areas were calculated for each image to determine the tumour vessel density using ImageJ software (National Institutes of Health).
10
Immunostaining of Tumor Tissue Sections
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Tumour tissues were fixed in 4% paraformaldehyde solution. The tissues were embedded in paraffin and cut into 2- or 6-µm sections. After heat-induced antigen retrieval in REAL Target Retrieval Solution (DAKO) and subsequent blocking of nonspecific sites with 0.1% normal goat serum/1% BSA, the tissues were immunostained with polyclonal anti-Ki67 antibody (Novus Biologicals, Littleton, CO, USA) and polyclonal anti-cleaved caspase-3 (Asp175) antibody (CST) diluted 1:200 followed by Alexa Fluor 594-conjugated goat anti-rat IgG staining. Sections were counterstained with DAPI and observed under a confocal laser scanning microscope (TCS SC8, Leica Microsystems, Wetzlar, Germany).
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