Next, GC and normal tissue paraffin sections were deparaffinized with xylene and rehydrated with gradient concentrations of ethanol. Microwave heating was used to retrieve antigens. Endogenous peroxidase was blocked by incubation in 0.3% H2O2. The slides were incubated with primary anti-CCNO antibody (1:1,000, ab47682, Abcam, Cambridge, UK) overnight and secondary antibody for 30 minutes. Finally, all slides were stained with 3,3′-diaminobenzidine and then counterstained with hematoxylin. Semiquantitative evaluation of IHC staining of CCNO was carried out using an immunoscore based on the percentage of stained cells and on staining intensity, as previously described. The intensity score was defined as follows: 0, no appreciable staining; 1, weak intensity; 2, moderate intensity; 3, strong intensity; 4, very strong intensity. The fraction score was based on the proportion of positively stained cells (0%–100%). The mean of the immunoscores from ten microscopic high-power fields was recorded. The histologic classification of the specimens was independently determined by two pathologists.
Ab47682
Manufactured by Abcam
Sourced in United Kingdom
Ab47682 is a laboratory antibody product. It is a purified polyclonal antibody that targets a specific protein. The core function of this product is to detect and bind to the target protein in biological samples.
Automatically generated - may contain errors
4 protocols using ab47682
1
CCNO Expression in Tissue Sections
2
Immunohistochemical detection of CCNO and RACK1
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The prepared paraffin sections were dewaxed and hydrated. After microwave antigen retrieval using 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), the sections were added with 3% H2O2-methanol. Next, the sections were added with primary antibody against CCNO (ab47682, 1:500, Rabbit, Abcam, Cambridge, UK), and RACK1 (5432, 1:1000, Rabbit, Cell Signaling Technologies, Beverly, MA, USA), and incubation was carried out overnight at 4 °C. Thereafter, the sections were re-probed with polymer enhancer (PV-9000, ZSGB-Bio, Beijing, China) at room temperature for 20 min. Next, the sections underwent further incubation with enzyme-labeled anti-mouse/rabbit polymer (PV-9000, ZSGB-Bio, Beijing, China) at room temperature for 30 min, and developed using 3,3′-diaminobenzidine (DAB) for 5 min. After the development was halted by distilled water, the sections were counterstained with hematoxylin, differentiated and blued. The sections were conventionally hydrated, cleared and sealed. Finally, the sections were photographed and observed under an inverted microscope (CX41, Olympus, Tokyo, Japan).
3
Immunohistochemical Staining of FFPE Samples
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Immunohistochemical staining of FFPE clinical samples was performed as described elsewhere20 (link). Polyclonal antibodies against CNTD2 (ab179781, Abcam) and CCNO (ab47682, Abcam) were used at a dilution of 1:100. For the analysis of ki-67 in tumours from xenografts, FFPE tissue sections were stained using the Dako Omnis (Dako, Agilent Technologies, Inc.) according to the manufacturer’s instructions and antigen retrieval was performed using EnVision FLEX Target Retrieval Solution, High pH (Dako, Agilent Technologies, Inc.). Tissue sections were subsequently incubated with primary antibody (GA626, Ki-67, clone MIB-1, ready-to-use, Dako, Agilent Technologies, Inc.) and the chromogenic visualization was performed using EnVision FLEX/HRP (Dako, Agilent Technologies, Inc.). Appropriate controls were stained concurrently to validate the procedure.
4
Immunohistochemical Analysis of CCNO
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OA and normal cartilage tissues were deparaffinized with xylene and rehydrated with gradient ethanol, followed by microwave antigen retrieval. The tissues were then immersed in 0.3% H2O2 to block the endogenous peroxidase and then immunostained with primary antibody against CCNO (1:1000, ab47682, Abcam) overnight. The following day, tissues were added with secondary antibody for 30 min of reaction. Finally, tissues were developed with 3,3-diaminobenzidine and counterstained with hematoxylin. Semiquantitative evaluation on the immunohistochemistry of CCNO was carried out using an immunoscore according to the percentage of stained cells and their staining intensity, as previously described. The intensity score was defined as follows: 0 = no evident staining; 1 = weak intensity; 2 = moderate intensity; 3 = strong intensity; 4 = very strong intensity. The fraction score was determined based on the proportion of positively stained cells (0–100%). The mean value of the immunoscores was recorded following observation under a microscope in ten randomly selected high-power fields. The histologic classification of the specimens was determined by two pathologists in an independent manner.
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