OV90 and A2780 cells or tissues were lysed using RIPA reagent (Thermo Fisher Scientific, Inc.) and the released total protein was extracted. The protein concentration was quantified using a BCA kit (Thermo Fisher Scientific, Inc.). A total of 10 µg protein sample/lane was separated via 10% SDS-PAGE, and the proteins were subsequently transferred to PVDF membranes (EMD Millipore). The membranes were incubated in 5% skimmed milk at room temperature for 2 h, then incubated with the corresponding primary antibody [β-catenin (cat. no. ab265591; 1:1,000; Abcam), cyclin D1 (cat. no. ab226977; 1:1,000; Abcam), c-myc (cat. no. ab152146; 1:1,000; Abcam) or GAPDH (cat. no. ab128915; 1:2,000; Abcam)] overnight at 4˚C. Subsequently, the washed membranes were incubated with the secondary antibody [HRP-labeled goat anti-rabbit (cat. no. ab205718; 1:5,000; Abcam)] at room temperature for 2 h. Bands were visualized by a Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.), and quantified using ImageJ version 1.51 (National Institutes of Health). β-actin was used as an internal reference.
Hrp labeled goat anti rabbit
Manufactured by Abcam
HRP-labeled goat anti-rabbit is a secondary antibody that is conjugated with horseradish peroxidase (HRP). It is used to detect and visualize primary antibodies raised in rabbit in various immunoassays and immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ripa reagent, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
C myc, by Abcam (1 mentions)
Cyclin d1, by Abcam (1 mentions)
Protease inhibitor, by Solarbio (1 mentions)
Anti igf2bp2, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
4 protocols using hrp labeled goat anti rabbit
1
Western Blot Analysis of Oncogenic Markers
2
Immunohistochemical Analysis of Ki-67 in Cancer Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cancer tissues from mice underwent a series of steps including fixation in 4% paraformaldehyde, dehydration, embedding in paraffin, and finally cutting into 5 µm slices. These slices were microwaved in citrate-buffered solution (pH 6.0) over 3 min for antigen retrieval. Following a 30 min block with 10% goat serum at room temperature, the sections and Ki-67 antibody (1:300, Ab15580) were co-incubated overnight at 4°C before the incubation with secondary antibody (HRP-labeled goat anti-rabbit, 1:1000, Abcam) for 30 min. The slices were exposed to DAB kit and stained with hematoxylin. The viewing of the images was carried out with the application of a light microscope (Leica Microsystems Inc., Illinois, USA).
3
Western Blot Analysis of Prostate Cancer Cell Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from prostate cancer cells was extracted using RIPA lysis buffer (cat. no. G2002; Wuhan Servicebio Technology Co., Ltd.) supplemented with protease inhibitors (Beijing Solarbio Science & Technology Co., Ltd.). After determining the concertation of each proteins using a BCA protein assay kit (cat. no. G2026; Wuhan Servicebio Technology Co., Ltd.), a total of 30 µg protein per lane was loaded on a 10% SDS-gel, resolved using SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 5% nonfat dry milk at room temperature for 2 h, and then incubated with anti-HMGCS1 (cat. no. ab155787; 1:500; Abcam), anti-IGF2BP2 (cat. no. ab117809; 1:500; Abcam) or anti-GAPDH (cat. no. ab9485; 1:500; Abcam) primary antibodies overnight at 4°C, and subsequently incubated with an HRP-labeled goat-anti-rabbit (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) secondary antibody for 2 h at room temperature. The bands were visualized using Immobilon™ Western Chemilum HRP Substrate (cat. no. WBKLS0100, MilliporeSigma) and analyzed using Quantity One software version 4.6.6 (Bio-Rad Laboratories, Inc.).
4
Immunohistochemistry Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For IHC, the slides were heated at 60˚C for 2 h, followed by the removal of paraffin using xylene. After being rinsed with ethanol, antigen recovery was conducted for 10 min and the samples were immersed in 3% hydrogen peroxide and subsequently blocked with 10% serum (cat. no. SL034; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 15 min. The slides were incubated with primary antibodies anti-CXCR4 (1:1,000; cat. no. ab181020) and anti-METTL3 (1:1,000; cat. no. ab195352; both from Abcam) at 4˚C overnight and then incubated with the corresponding secondary antibody, HRP-labeled goat anti-rabbit (1:2,000) for 1 h at room temperature. The slides were then stained with DAB work solution (cat. no. P0202; Beyotime Institute of Biotechnology) at room temperature, counterstained with hematoxylin work solution (cat. no. C0107; Beyotime Institute of Biotechnology) at room temperature and identified with 1% hydrochloric acid alcohol. Finally, slides were fixed with neutral balsam (cat. no. G8590; Solarbio Science & Technology Co., Ltd.) at room temperature and visualized under a light microscope (magnification, x20; Nikon Corporation).
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!