Paraffin sections of xenograft tumor tissues were cut with a thickness of 4 μm. Sections were incubated with primary antibodies: anti-RTKN2 (Abcam, #ab251807, 1:1000), anti-β-catenin (Abcam, #ab16051, 1:1000), anti-Ki67 (Abcam, #ab245113, 1:1000), anti-E-cadherin (Abcam, #ab1416, 1:500), and anti-N-cadherin (Abcam, #ab76057, 1:1000). The temperature was maintained at 4 °C overnight. Sections were co-incubated with HRP‐polymer‐conjugated secondary antibodies after washing with phosphate‐buffered saline, then, they were immunostained using DAB plus kit.
Ab245113
Manufactured by Abcam
Sourced in United Kingdom
Ab245113 is a laboratory reagent product. It is a monoclonal antibody designed for research use. The core function of this product is to serve as a detection and/or quantification tool in scientific experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ab92552, by Abcam (3 mentions)
Ab1416, by Abcam (2 mentions)
Ab16051, by Abcam (2 mentions)
Ab76057, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab181603, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ab81083, by Abcam (2 mentions)
Chemiluminescence detection kit, by Beyotime (2 mentions)
Ripa solution, by GenePharma (2 mentions)
Ab150113, by Abcam (2 mentions)
Ab251807, by Abcam (1 mentions)
Ab32158, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab32370, by Abcam (1 mentions)
Dmi3000b, by Leica (1 mentions)
Ab89901, by Abcam (1 mentions)
Ab236466, by Abcam (1 mentions)
Ab110640, by Abcam (1 mentions)
13 protocols using ab245113
1
Immunohistochemical Analysis of Xenograft Tumors
2
Immunohistochemistry Protocol for Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protocol of IHC was described previously [36 (link)]. Sections were prepared in 10 μm. The primary antibodies against β-catenin (Abcam, #ab16051, 1:1000), Ki67 (Abcam, #ab245113, 1:1000), PDGFD, E-cadherin (Abcam, #ab1416, 1:500), and N-cadherin (Abcam, #ab76057, 1:1000) were used. Following PBS washes, secondary antibodies (1:1000 was applied to sections. Lastly, the signal was developed by DAB and validated by two experienced pathologists.
3
Immunohistochemical Analysis of Ki67 in Mice Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We obtained the mice tumor tissues, which were fixed by using the 10% (v/v) formaldehyde and the tissues were subsequently embedded into the paraffin. Then, the above tissues were prepared as sections with about 5 μm thickness. After that, according to the protocols provided by the previous publication [34 (link)], we performed IHC assay to examine the expression status, including expression levels and localization of Ki67 protein in mice tumor tissues. Briefly, the tissues were sequentially incubated with primary antibody against Ki67 (1:400, #ab245113, Abcam, UK) and secondary antibody (Cell signaling Technology, USA), and the yellow Ki67-postive cells were observed and counted under a light microscope (ThermoFisher Scientific, USA).
4
Immunohistochemical Analysis of Tumor Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess the expression of proliferation-related protein (Ki67), apoptosis-related proteins (BAX, BAK, BIM, BCL-2, BCL-XL and MCL1) and metastasis-associated protein (MMP9) in tumor tissues, immunohistochemistry assay was conducted with the following antibodies as previously reported [27 (link)]. Anti-Ki67 (ab245113), anti-BAX (ab32503), anti-BAK (ab32371), anti-BIM (ab32158), anti-BCL-2 (ab32124), anti-BCL-XL (ab32370), anti-MCL1 (ab32087) and anti-MMP9 (ab76003) were purchased from Abcam.
5
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein in cells was extracted by RIPA solution (GenePharma) and the protein concentration was estimated through BCA Protein Assay Kit (Beyotime). Equal amounts protein samples were loaded on each lane and separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Next, the membrane were cultivated with primary antibodies against PCNA (ab92552, 1:1,000 dilution, abcam), Ki-67 (ab245113, 1:10,000 dilution, abcam), Bax (ab81083, 1:1,000 dilution, abcam), Caspase-3 (218161, 1:1000 dilution, abcam) and GAPDH (ab181603, 1:1,000 dilution, abcam) at 4°C overnight. The membrane was washed with PBST for three times. Then the membrane was incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721, 1:1,000 dilution, abcam). The bands were visualized using a chemiluminescence detection kit (Beyotime).
6
Quantifying Ki67 Expression in Tumor Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tumors from nude mice were fixed, embedded, and sectioned (5 μm). IHC staining was performed as instructed by the kit (Abcam). After overnight incubation with the primary antibody Ki67 (1:200, ab245113, Abcam), the tumor sections were incubated with the secondary antibody goat anti-mouse IgG H&L (Alexa Fluor® 488) (ab150113, Abcam) for 30 min. This step was followed by the addition of 3,3-diaminobenzidine H2O2 solution (Zhongshan Golden Bridge, Beijing, China) for development, and counterstaining with hematoxylin. Five images of the regions of interest without overlapping were taken and the positive cells per image were quantified using Image-Pro Plus 6.0 software. The results were presented as the average percentage of positive cells per image. The group without the primary antibody served as the negative control group.
7
Immunohistochemical Analysis of Ovarian Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytospins were prepared with cellularized tumor cells for immunohistochemical studies, as described previously.28 Then, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min, and blocked with 5% normal goat serum for 1 h at room temperature. The cytospins were then incubated at 4°C overnight with primary rabbit/mouse monoclonal antibodies generated against the following proteins: WT1, a protein that is expressed at high levels in patients with epithelial ovarian cancer (1:600, ab89901, Abcam, Cambridge, UK); MUC16, a transmembrane glycoprotein expressed at high levels in over 80% of patients with ovarian cancer; CA125 (1:2000, ab110640, Abcam); and Ki67, as an indicator of tumor proliferation (1:400, ab245113, Abcam). Subsequently, a goat anti-rabbit secondary antibody (ab236466, Abcam) was applied for 10 min at room temperature; a mouse-specific reagent was incubated for 10 min prior to this step if a mouse primary antibody had been applied. Immunoreactive signals were visualized by incubation in diaminobenzidine (DAB) for 5 min. Finally, we captured images with a microscope (Leica DMI3000 B, Germany) and edited them with Photoshop (Adobe, USA).
8
Western Blot Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TLysis buffer (Cell Signaling Technology, Danfoss, MA, USA) containing phenylmethanesulfonyl fluoride (Beyotime) was used to lyse the stimulated cancer cell lines. Equal amounts of extracts were separated to 10% or 12.5% SDS PAGE gels (Abcam, Cambridge, UK), transferred onto polyvinylidene fluoride membranes (EMD Millipore), followed by incubation with primary antibodies at 4°C overnight. The primary antibodies were anti-CDC6 (ab109315, abcam), Ki-67 (ab245113, abcam), anti-PCNA (ab92552, abcam) and GAPDH (AF0009; Beyotime). HRP-conjugated secondary antibodies were used to incubate the membranes for 1 hour at room temperature, after which they were washed with TBST (PBS with 0.05% Tween20) 6 times. Finally, the visualization of the blot bands was achieved by Find-do ×6 Tanon (Tanon, Shanghai, China).
9
Immunohistochemical Analysis of Ki67 in Tumor Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tumor tissues were fixed with 4% formaldehyde solution, embedded in paraffin, and cut into 4 µm thickness. Subsequently, H&E staining and immunohistochemical staining were presented. For H&E staining, the sections were stained with hematoxylin for 5 min and stained with eosin staining solution for 3 min. For immunohistochemical staining, the sections were incubated with mouse anti-human Ki67 monoclonal antibody (ab245113, Abcam, Cambridge Science Park, CK) at 4 °C overnight. The sections were then incubated with HRP-labeled Goat Anti-Mouse IgG (ab150113, Abcam) at 37 °C for 20 min. Hematoxylin staining solution was used to stain the nuclei. After ethanol dehydration and xylene transparency, the sections were sealed with neutral gum. The stained sections were observed under an optical microscope (400×).
10
Paraffin IHC for Ki67 quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemistry (IHC) of paraffin sections, endogenous peroxide activity was quenched through 10 min incubation in 3% H2O2, after which non-specific binding was blocked with serum. The resulting sections were incubated with an anti-Ki67 (Abcam ab245113) antibody at a 1:100 dilution overnight. Subsequently, diluted biotinylated anti-rabbit IgG (Vectastain kit) was added to the sections and incubated for 30 min. Next, Vectastain ABC reagent (Vector) and 3, 30-diaminobenzamidine (DAB) were used for color development. Counterstaining was performed using hematoxylin solution.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!