Total protein was extracted by RIPA lysis buffer (Keygen). After detection of protein concentration, extracted protein was separated with SDS‐PAGE (Beyotime). After transferring onto PVDF membranes (Millipore), the membranes were blocked in non‐fat dried milk (Yili). After that, the membranes were probed with unique primary antibodies: KI67 (ab181255; 1:5000; Abcam), Bax (ab32503; 1:3000; Abcam), multidrug resistance protein (MRP1; ab76464; 1:1000; Abcam), CRABP2 (ab181255; 1:3000; Abcam), and β‐actin (ab8226; 1:1000; Abcam), and then secondary antibody was used for combination with primary antibodies. At last, the combined signals were detected by ECL reagent (Abcam).
Ab181255
Manufactured by Abcam
Ab181255 is a lab equipment product. It is a primary antibody that specifically recognizes a target protein. The core function of this product is to identify and bind to the target protein for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ab76464, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ecl reagent, by Abcam (1 mentions)
Sds page, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Keygen Biotech (1 mentions)
Ab171870, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Dako real detection kit, by Agilent Technologies (1 mentions)
Autostainer link 48, by Agilent Technologies (1 mentions)
Ab51608, by Abcam (1 mentions)
4 protocols using ab181255
1
Protein Expression Analysis Protocol
2
Immunohistochemical Staining of Tumor Tissues
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Tumor tissues were fixed in formalin solution (10%) and embedded in paraffin, followed by cutting into 4‐μm‐thick section. Afterwards, these sections were probed with the primary antibody: CRABP2 (ab181255; 1:100; Abcam) or Ki67 (ab15580; 1:200; Abcam). Next, these sections were incubated with secondary antibody (ab171870; 1:2000; Abcam). After staining with diaminoaniline (DAB; Maxim, Fuzhou, China) and counterstaining with hematoxylin (Maxim), the sections were observed using a microscopy (Leica).
3
MPNST Immunohistochemistry in NF1 Patients
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Human MPNST specimens from NF1 patients and non-NF1 patients were kindly provided by Prof. W. Paulus (Institute of Neuropathology, University Hospital Muenster, Germany). These experiments were approved by ethics committee at „Ärztekammer Westfalen-Lippe Münster”(2007-261-f-S). Written informed consent was provided for participation. MPNST cell pellets were frozen at -80°C, fixed in 4% PFA and stored in 70% ethanol until paraffin embedding. Hematoxylin and eosin staining (HE) of FFPE samples was performed in an automated staining instrument Tissue Tek Glas and Prisma system (Sakura). For immunohistochemistry of FFPE samples, slices were deparaffinised and pre-treated with target retrieval solution (pH 6.1). Immunohistochemistry was performed using the DAKO REAL™ Detection Kit (K5001) according to the provided protocol and the automated staining instrument AutostainerLink 48 (Dako). Deparaffinised sections were incubated with primary anti-CRABP2 antibody (Abcam ab181255, monoclonal, from rabbit, 1:100). Staining was visualized by DAB staining followed by nuclear counterstaining with hematoxylin.
4
CRABP2 and HIF1α Immunohistochemistry
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In brief, tissue sections were firstly deparaffinized and graded with xylene and alcohol solutions, and then exposed antigen by high pressure heat repair in 10 mM citrate buffer (pH 6.0) and blocked endogenous peroxidase by 3% hydrogen peroxide solution. Then sections were incubated with primary antibody CRABP2 (1:100, Cat#ab181255, abcam) or HIF1α (1:100, Cat#ab51608, abcam) for overnight at 4°C, and incubated with secondary antibody for 1 h at 37°C following with DAB staining for 5 min to visualize immunolabeling. The expression levels of CRABP2 or HIF1α were obtained by multiplying the percentage of positive staining score with the staining intensity score. Percentage points is defined as: 1 (0% ~ 25%), 2 (26% ~ 50%), 3 (51% ~ 75%), 4 (76% ~ 100%). Intensity scores were defined as: 0 (no staining), 1 (low staining), 2 (moderate staining), and 3 (high staining). According to the final score, all tissues were divided into high expression group (score ≥6) and low expression group (score < 6).
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