Cells were seeded in four-well plastic dishes to measure cell proliferation. For each condition, cells were washed twice with PBS and fixed with 4% formaldehyde in 0.5 ml PBS for 20 min at room temperature. Then, the cells were washed again with PBS. Next, the cells were blocked in PBS containing 0.5% BSA and incubated for 1 h with polyclonal Ki-67 antibody (Millipore). Then, the cells were washed three times for 10 min with PBS and incubated with FITC-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) secondary antibody for 1 h. All images were visualized by confocal microscopy (LSM 710; Zeiss), and the acquired images were transferred to a computer equipped with Zen Light Edition software (Zeiss).
Ki67 antibody
Manufactured by Merck Group
Sourced in United States
The Ki67 antibody is a laboratory tool used for the detection and quantification of the Ki-67 protein, which is a marker of cellular proliferation. The Ki67 antibody is commonly used in immunohistochemistry and flow cytometry applications to assess cell cycle status and tissue proliferation rates.
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Lab products found in correlation
P38δ antibody, by R&D Systems (2 mentions)
Phospho fak tyr397, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Phospho stat3 ser727, by Cell Signaling Technology (2 mentions)
Phospho p38, by Cell Signaling Technology (2 mentions)
Phospho erk1 2, by Cell Signaling Technology (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
β actin antibody, by Merck Group (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Fitc conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Zen light edition software, by Zeiss (1 mentions)
Triton x, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
4 6 diamino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Occludin antibody, by Abcam (1 mentions)
Primer synthesis, by Takara Bio (1 mentions)
Phospho nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
Nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
12 protocols using ki67 antibody
1
Cell Proliferation Assay with Ki-67
2
Western Blot Antibody Procurement
3
Western Blot Antibody Procurement
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p38δ antibody was purchased from R&D systems. FAK, phospho-FAK Tyr397, p38s (α, β and γ), phospho-p38s, Cyclin D1, ERK1/2, phospho-ERK1/2, Stat3 and phospho-Stat3 Ser727 antibodies were obtained from Cell Signaling Technology. β-actin antibody was purchased from Sigma-Aldrich. Ki67 antibody was obtained from Millipore. When molecular weight did not overlap, same western blott membranes were re-used for different antibodies after stripping the previous antibody.
4
Immunostaining of Breast Cancer Tissues
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Fresh specimens of the primary breast cancer tissues and tamoxifen-resistant breast cancer tissues were collected from Wuzhong People's Hospital. All patients signed a written informed consent, and the present study was approved by the Ethics Committee of Wuzhong People's Hospital, Suzhou, China. The clinical data of patients are presented in Table I . A total of 40 samples were collected and divided into two groups, with 20 samples in each group. The tumor specimens were fixed by 4% neutral formalin, embedded in paraffin and sectioned into 4-µm thick sections by standard SP method. Immunostaining was performed with the avidin-biotin-peroxidase complex method (Ultrasensitive™; MaiXin). Then tissue sections were incubated with TCRP1 antibody (cat. no. HPA037580; rabbit; 1:200 Sigma-Aldrich; Merck KGaA), SGK1 antibody (cat. no. S5188; rabbit, 1:2,000; Sigma-Aldrich; Merck KGaA), Ki67 antibody (cat. no. 9129; rabbit; 1:400; Cell Signaling Technology, Inc.) overnight at 4°C, followed by incubation with a goat anti-rabbit biotinylated secondary antibody (cat. no. SA1020; dilution 1:1,000; Boster Bio) at 4°C overnight. The images were analyzed with IPLab 4.0 imaging software (Scanalytics, Inc.).
5
Evaluating MTX's Impact on Cell Division
6
Traditional Chinese Medicine Enema for Colitis
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DSS (AppliChem, Lot#：A3261‐0250 Germany); the six medicinal herbs of QBD raw powder, as shown in Table 1 (Pharmacy Department, The Second Hospital affiliated to Shanxi University of TCM, Xianyang, China) were decoting up 150 mL by traditional decocting method for enema； Mesalazine Enemas (V Vifor AG Zweigniederlassung Medichemie Ettingen， H20150127, Switzerland); faecal occult blood (FOB) (Baso Diagnostics Inc， Zhuhai， china); NF‐κB p65 antibody (Cell Signaling, #8242, Danvers, MA, USA); Phospho‐NF‐κB p65 antibody (Cell Signaling, #3039); p44/42 MAPK (ERK1/2) (antibody (Cell Signaling，#4695); Phospho‐p44/42 MAPK (ERK1/2) antibody (Cell Signaling, #4370); MMP‐9 antibody (Merck, AB19016, Burlington, MA, USA); Ki67 antibody (Merck, AB9260); Cleaved Caspase‐3 antibody (Cell Signaling, #9664); Caspase‐3 antibody (Cell Signaling, #9662); β‐Actin antibody (Cell Signaling,# 4970) Notch1 antibody (Cell Signaling, #3608s); ZO‐1 antibody (abcam, ab96587, Cambridge, MA, USA); Occludin antibody (abcam, ab 21632); claudin‐1 antibody (abcam, ab15098); primer synthesis (Takara Bio Inc, Dalian, China).
7
Immunofluorescence Staining for Ki67 Expression
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Cells were cultured in 24 well plates and fixed with 4%
paraformaldehyde. After rinsing with phosphate buffered
solutions (PBS, Invitrogen, USA) the samples were
permeabilized with 0.1% Triton (Invitrogen, USA)/PBS
and blocked with 1% bovine serum albumin (BSA, Sigma
Aldrich)/PBS. After removing the blocking solution, the cellswere incubated overnight with the primary Ki67 antibody(Sigma Aldrich, USA). After rinsing, the process was followed
by incubation with species-specific secondary antibodies,
which were conjugated with fluorochrome; the labeledcells were counterstained with 0.2 µg/ml 4’, 6-diamidino2-
phenylindole (DAPI, DAPI, Sigma Aldrich, USA) for 3minutes at room temperature and fixed with Mowiol 4-88
reagent (Merck, USA). Labeled cells were examined with aconfocal microscope Zeiss LSM 700, and images were taken
with a Zeiss LSM-TPMT camera (6 (link)).
paraformaldehyde. After rinsing with phosphate buffered
solutions (PBS, Invitrogen, USA) the samples were
permeabilized with 0.1% Triton (Invitrogen, USA)/PBS
and blocked with 1% bovine serum albumin (BSA, Sigma
Aldrich)/PBS. After removing the blocking solution, the cellswere incubated overnight with the primary Ki67 antibody(Sigma Aldrich, USA). After rinsing, the process was followed
by incubation with species-specific secondary antibodies,
which were conjugated with fluorochrome; the labeledcells were counterstained with 0.2 µg/ml 4’, 6-diamidino2-
phenylindole (DAPI, DAPI, Sigma Aldrich, USA) for 3minutes at room temperature and fixed with Mowiol 4-88
reagent (Merck, USA). Labeled cells were examined with aconfocal microscope Zeiss LSM 700, and images were taken
with a Zeiss LSM-TPMT camera (6 (link)).
8
Ki67 Immunostaining Protocol
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Immunostaining of Ki67 were carried out as previously described 23 (link). Briefly, sections were incubated with Ki67 antibody (1:200, Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. Horseradish peroxidase (HRP)-conjugated secondary antibodies were further incubated and visualized with 3, 3'-diaminobenzidine (DAB).
9
Quantifying Aortic Wall Remodeling and VSMC Characteristics
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Tissue sections were stained by H&E staining and observed under the Leica DM3000 microscope (Leica, Germany) to measure aortic wall thickness (WT), wall cross-sectional area (WCSA), lumen diameter (ID), and wall/lumen ratio. Fibers and collagens were stained by Masson's trichrome staining and Sirius-red staining. Quantitative analyses were performed by ImageJ.
After deparaffinage, rehydration, and permeabilization in 1‰ Triton X-100, double-immunofluorescence staining was performed on paraffin sections. The VSMCs in the aorta were labeled by anti-α-SMA antibody (Sigma Aldrich, USA). The ratio of NOX4-positive VSMCs and the expression of SOD2 in aortic VSMCs were marked with anti-NOX4 antibody and anti-SOD2 antibody (Sigma Aldrich, USA), respectively. Proliferative VSMCs were labeled with anti-PCNA antibody (Sigma Aldrich, USA) and Ki67 antibody (Sigma Aldrich, USA), respectively.
After deparaffinage, rehydration, and permeabilization in 1‰ Triton X-100, double-immunofluorescence staining was performed on paraffin sections. The VSMCs in the aorta were labeled by anti-α-SMA antibody (Sigma Aldrich, USA). The ratio of NOX4-positive VSMCs and the expression of SOD2 in aortic VSMCs were marked with anti-NOX4 antibody and anti-SOD2 antibody (Sigma Aldrich, USA), respectively. Proliferative VSMCs were labeled with anti-PCNA antibody (Sigma Aldrich, USA) and Ki67 antibody (Sigma Aldrich, USA), respectively.
10
Immunofluorescence Analysis of Breast Cell Lines
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