After fixing, embedding, sliding, and deparaffinizing, tissue sections were blocked with 3% H2O2 and 5% BSA. Then sections were incubated with anti-FOXD1(Invitrogen, Cat No. PA5-35145), anti-KI67 (Proteintech, Cat No. 27309-1-AP), anti-PCNA (Proteintech, Cat No. 10205-2-AP), and anti-GLUT1(abcam, Cat No. ab115730) antibodies at 4 °C overnight. After washing with PBS, immunohistochemical secondary antibody was applied to the sections for 1 h at room temperature, followed by DAB staining, hematoxylin re-staining, and imaging. The results were evaluated blindly by two independent pathologists.
Anti ki67
Manufactured by Proteintech
Sourced in United States, China
Anti-Ki67 is a laboratory equipment product that is used to detect the presence of the Ki67 protein, a widely used marker for cell proliferation. The core function of Anti-Ki67 is to enable the identification and quantification of Ki67-positive cells in tissue samples.
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Lab products found in correlation
Anti pcna, by Proteintech (10 mentions)
Anti e cadherin, by Proteintech (4 mentions)
Anti glut1, by Abcam (3 mentions)
Anti vimentin, by Proteintech (3 mentions)
Anti n cadherin, by Proteintech (3 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti mettl1, by Proteintech (2 mentions)
Anti cyclin d1, by Proteintech (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti cd31, by Proteintech (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Axio scope a1 microscope, by Zeiss (2 mentions)
Anti ldha, by Proteintech (2 mentions)
Anti hk2, by Abcam (2 mentions)
Anti hif 1α, by Abcam (2 mentions)
Sds page gel, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by OriGene (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Bca protein assay kit, by Solarbio (2 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (2 mentions)
59 protocols using anti ki67
1
Immunohistochemical Analysis of FOXD1, KI67, PCNA, and GLUT1
2
Antibody-based Expression Analysis in Cancer Cells
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The following antibodies were used for immunofluorescence staining: anti-GAPDH (1:5,000; cat. no. Ab8245; Abcam); anti-UCP2 (1:100; cat. no. 11081-1-AP; ProteinTech Group, Inc.) and anti-FLNa (1:100; cat. no. ab76289; Abcam). The antibodies were used to detect UCP2 and FLNa expression in different CC cell lines.
The following antibodies were used for immunohistochemical staining: anti-UCP2 (1:200; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:200; cat. no. ab76289; Abcam), anti-P16 (1:200; cat. no. 10883-1-AP; ProteinTech Group, Inc.), and anti-Ki67 (1:1,000; cat. no. ab92742; Abcam).
The following primary antibodies were used for western blotting of relevant proteins: anti-UCP2 (1:1,000; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:1,000; cat. no. ab76289; Abcam), anti-GAPDH (1:5,000; cat. no. ab8245; Abcam), anti-ERK1/2 (1:1,000; cat. no. 4695; Cell Signaling Technology, Inc.), anti-P-ERK1/2 (1:1,000; cat. no. 4370; Cell Signaling Technology, Inc.), anti-AKT1 (1:1,000; cat. no. ab227100; Abcam), anti-P-AKT1 (1:10,000; cat. no. ab81283; Abcam), and anti-BCL-2 (1:1,000 cat. no. ab32124; Abcam).
The following antibodies were used for immunohistochemical staining: anti-UCP2 (1:200; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:200; cat. no. ab76289; Abcam), anti-P16 (1:200; cat. no. 10883-1-AP; ProteinTech Group, Inc.), and anti-Ki67 (1:1,000; cat. no. ab92742; Abcam).
The following primary antibodies were used for western blotting of relevant proteins: anti-UCP2 (1:1,000; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:1,000; cat. no. ab76289; Abcam), anti-GAPDH (1:5,000; cat. no. ab8245; Abcam), anti-ERK1/2 (1:1,000; cat. no. 4695; Cell Signaling Technology, Inc.), anti-P-ERK1/2 (1:1,000; cat. no. 4370; Cell Signaling Technology, Inc.), anti-AKT1 (1:1,000; cat. no. ab227100; Abcam), anti-P-AKT1 (1:10,000; cat. no. ab81283; Abcam), and anti-BCL-2 (1:1,000 cat. no. ab32124; Abcam).
3
Western Blot Analysis of Protein Expression
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Whole protein was extracted from transfected cells after cells were lysed using the RIPA buffer containing PMSF and phosphatase inhibitors. After quantifying by the BCA Protein Assay Kit (Beyotime, Shanghai, China), the proteins were resolved by using 10% sodium dodecyl SDS-PAGE, and then proteins were transferred to PVDF membranes. For clearer western blot bands, the blots were cut prior to hybridisation with antibodies. Membranes were incubated with specific primary antibodies ( anti-TUBA1C (Proteintech, Wuhan, China), anti-E2F1 (Proteintech), anti-ki-67 (Proteintech), anti-PCNA (Proteintech), GAPDH (Proteintech), and β-actin (Proteintech)) diluted in antibody diluent at 4 °C for 12 h. Then incubating the membranes with a secondary antibody for 2 h. The western blot bands were scanned by Amersham Imager 600 and analyzed by Image J software.
4
Immunohistochemical Evaluation of RPS24 and Ki67
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The operation process for the immunohistochemistry (IHC) analysis is referred to in our previous report [45 (link)]. The primary antibodies were anti-RPS24 (rabbit, 1:100, ABclonal Technology, Wuhan, China, A12123) and anti-Ki67 (rabbit, 1:7000, Proteintech, Wuhan, China 14831-1-AP).
The IHC scores were assessed independently by three pathologists. The staining area of RPS24 was quantified according to the proportion of stain-positive cells: 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The staining intensity scores consisted of 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong). The final staining scores were calculated using the intensity and percentage scores. Finally, tissue scores of ≥4 were defined as high expression, while the others were defined as low expression.
The IHC scores were assessed independently by three pathologists. The staining area of RPS24 was quantified according to the proportion of stain-positive cells: 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The staining intensity scores consisted of 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong). The final staining scores were calculated using the intensity and percentage scores. Finally, tissue scores of ≥4 were defined as high expression, while the others were defined as low expression.
5
Immunohistochemical Analysis of POLA2 and Ki-67 in Tumor Tissue
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Tumor tissue was paraffin-embedded and cut into 4-µm cross-sections. After dewaxing, antigen repair and blocking, sections were incubated with anti-POLA2 (Proteintech, Wuhan, China) and anti-ki-67 (Proteintech, Wuhan, China) for 2 h at 37 °C. The tissue was then incubated with biotin-labeled goat anti-rabbit secondary antibody for 20 min, followed by incubation with HRP-labeled streptavidin for 10 min at 37 °C. After washing with PBS, the nuclei were stained using hematoxylin solution.
6
Immunohistochemistry for Protein Expression
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Immunohistochemistry staining was performed as previously described45 (link). The following primary antibodies (anti-METTL1, Proteintech, 1:2000 dilution; Anti-WDR4, Abcam, 1:1000 dilution; Anti-Ki67, Proteintech, 1:8000 dilution; Anti-RPTOR, Proteintech, 1:500 dilution; Anti-pS6K1, Cell signaling technology, 1:1000 dilution; Anti-pULK1, Affinity, 1:1000 dilution; Anti-p4EBP1, Cell signaling technology, 1:1000 dilution) were used for detection of indicated protein expression. IHC staining was evaluated as H-score using QuPath (v0.2.3) by recording the staining intensity and the proportion of the cells with positive staining46 (link). Tissues with H-score of ≧100 were defined as high expression samples, and tissues with H-score below 100 were defined as low expression samples. For survival analysis, patients were stratified into four groups based on their relative levels of H-scores: (>75%, 50–75%, 25–50%, and 0–25%).
7
Immunohistochemical Analysis of Oncogenic Markers
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Immunohistochemistry (IHC) staining was performed as previously described (23 (link)). The primary antibodies included anti-PKCι (Abcam, Inc., Cambridge, UK), anti-Smoothened (Abcam, Inc., Cambridge, UK), anti-GLI1 (Abcam, Inc., Cambridge, UK), anti-HHAT (Novus Biologicals, Littleton, CO, USA), and anti-Ki-67 (ProteinTech, Chicago, IL, USA). The secondary antibodies included anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA). The proportion of PRKCI, SMO, and GLI1 staining was scored as follows: less than 1/3 = 1, between 1/3 and 2/3 = 2, or more than 2/3 = 3. The staining intensity was scored as follows: negative = 0, light yellow = 1, brownish yellow = 2, or tan = 3. The final score for PRKCI, SMO, and GLI1 expression was calculated by multiplying the 2 scores. The slides were classified into low- and high-expression groups, corresponding to scores of <6 and ≥6, respectively. The histopathological diagnosis of the patients included in our study was established by two pathologists who specialized in gynecologic oncology.
8
Protein Expression Analysis via Western Blot
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The proteins were isolated through using SDS‐PAGE, and then the separated proteins were transferred onto PVDF membrane. The membrane was incubated with primary antibodies, anti‐KRT5 (1:2000, Proteintech, Chicago, USA), anti‐GAPDH (1:1000, Proteintech), anti‐Ki67 (1:1500, Proteintech), anti‐Twist1 (1:2000, Proteintech), and anti‐E‐cadherin (1:2000, Proteintech) at 4 °C overnight. The blots were exposed using enhanced chemiluminescence (ECL) system (Thermo Fisher).
9
Immunohistochemical Profiling of Mouse Liver and Spleen
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Paraffin-embedded, formalin-fixed tissues of mice liver and spleen were immunostained for Ki67, CK7 and CK20 proteins. The signal was amplified and visualized with diaminobenzidine-chromogen, followed by counterstaining with hematoxylin. Anti-Ki67 (1:50), anti-CK7 (1:50) and anti-CK20 (1:50) were purchased from Proteintech (CK7 and CK20) and Bioworld (Ki-67).
10
Immunohistochemical Antibody Evaluation
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IHC and its evaluation were carried out as previously reported [13 (link)]. The rabbit anti-ING5, anti-ki-67 and anti-LC-3B antibodies were purchased from Proteintech, DAKO and Santa Cruz respectively.
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