IF staining was performed based on published methods (39 (link), 40 (link)). Primary antibodies were used at 1:150 dilution as following: EYA2 (ab95875, Abcam), YBX1 (sc-101198, Santa Cruz), EGFR (sc-03, Santa Cruz) and PCNA (sc-7907, Santa Cruz). The goat anti-mouse and the goat anti-rabbit secondary antibodies (Alexa Fluor-568) were both used at 1:300. Cell nuclei were stained with Hoechst 33342 at the dilution of 1:1,000.
Ab95875
Manufactured by Abcam
Ab95875 is a high-quality antibody product manufactured by Abcam. It is designed for use in various laboratory applications, including immunoassays and cell-based studies. The product details and specific applications are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Sc 03, by Santa Cruz Biotechnology (1 mentions)
Sc 7907, by Santa Cruz Biotechnology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Envision flex target retrieval solution, by Agilent Technologies (1 mentions)
Envision flex system, by Agilent Technologies (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
P akt clone d9e, by Cell Signaling Technology (1 mentions)
Ab31392, by Abcam (1 mentions)
Fluorchem fc2 imaging system, by Bio-Techne (1 mentions)
Sc 32233, by Santa Cruz Biotechnology (1 mentions)
Hbre duc140sur 01, by Shanghai Outdo Biotech Co. (1 mentions)
4 protocols using ab95875
1
Immunofluorescence Staining Protocol
2
Immunohistochemical Analysis of EYA2, PTEN, and p-AKT
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The FFPE sections were immunostained using the Dako EnVision™ Flex+ System (K8012; Dako, Glostrup, Denmark). Deparaffinization and epitope unmasking were carried out in a PT-Link using an EnVision™ Flex target retrieval solution (Dako, Carpinteria, CA, USA). The sections were treated with 0.3% hydrogen peroxide (H2O2) for 5 min to block endogenous peroxidase. Sections were incubated overnight at 4°C with the following antibodies: EYA2 (ab95875, Abcam), PTEN (ab31392, Abcam) and p-AKT (clone D9E, Cell Signaling Technology). The specimens were subsequently treated with EnVision™ Flex linker mouse or rabbit (15 min), EnVision™ Flex/HRP enzyme (30 min), and 3′3-diaminobenzidine tetrahydrochloride (10 min). The samples were counterstained with hematoxylin, dehydrated and mounted on a Richard-Allan Scientific Cyto seal XYL (Thermo Scientific, Waltham, MA, USA). The protein expression was scored semi-quantitatively based on the percentage of positive cells utilizing the following scale: +, <25%; ++, 25–49%; +++, 50–74%; and ++++, 75–100%.
3
Western Blot Analysis of EYA2, PTEN, and GAPDH
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Western blot analysis was performed as previously described [58 ]. Briefly, cells were lysed in cold lysis buffer, proteins (20–30 μg) were resolved on SDS-PAGE, transferred onto PVDF membranes, and probed with antibodies for EYA2 (ab95875, Abcam), PTEN (ab31392, Abcam), and GAPDH (sc-32233, Santa Cruz Biotechnology) at 4°C overnight. Detection was performed with the SuperSignal West Femto Maximum Sensitivity Substrate Trial Kit (Pierce, Rockford, IL, USA). The band images were digitally captured and quantified with a FluorChem FC2 imaging system (Alpha Innotech, San Leandro, CA, USA). Protein levels were normalized to GAPDH and quantified with respect to vector control group.
4
Evaluating EYA2 Protein in Breast Cancer
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One commercially available TMA slide (HBre-Duc140Sur-01, Shanghai Outdo Biotech Co., Ltd.) was purchased for IHC analysis, which contained histologically confirmed breast cancer tissues with clinico-pathological information, such as tumor grade, clinical stage and the status of ER, PR, and HER2 in IHC (Table 1 ). Breast tumors with positive status of ER or PR belong to luminal-type, and tumors that do not express ER, PR, and HER2 are TNBC. Due to tissue shedding of 15 cases, the number of actually available tissue points was 125. To evaluate the protein abundance of EYA2 in ER– vs. ER+, PR– vs. PR+, and luminal-type vs. TNBC tissues as well as the prognostic value among breast cancer population, IHC analysis was conducted with a standard protocol described previously (23 (link)). The specific primary antibody against EYA2 (ab95875, Abcam) was utilized for IHC at a dilution of 1:100.
Two experienced pathologists performed IHC scoring independently with no prior knowledge of the clinico-pathological information. The multiplication of intensity and proportion of positive-staining tumor cells was exploited to quantify the protein levels of EYA2 according to a standard protocol as described previously (24 (link)).
Two experienced pathologists performed IHC scoring independently with no prior knowledge of the clinico-pathological information. The multiplication of intensity and proportion of positive-staining tumor cells was exploited to quantify the protein levels of EYA2 according to a standard protocol as described previously (24 (link)).
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