Using a microtome (Leica), 8 µm thick slices were taken from formalin-fixed paraffin-embedded (FFPE) prostate tissue blocks and mounted on Tomo microscope slides (Matsunami, Bellingham, WA, USA). Slides were then stained for IGF-II peptide using an IGF-II antibody (AbCam, Cambridge, UK) at a dilution of 1:600, on a Ventana BenchMark ULTRA™ machine (Roche, Oro Valley, AZ, USA). Slides were scored by two pathologists using a modified version of the Allred system, combining the proportion of tissue stained (a scale of 1–5) with the staining intensity (a scale of 1–3) to give a score out of 8 [55 ]. Currently, there is no standardized method of scoring prostate tissue.
Igf 2 antibody
Manufactured by Abcam
Sourced in United Kingdom
The IGF-II antibody is a laboratory reagent used for the detection and quantification of Insulin-like Growth Factor II (IGF-II) in biological samples. IGF-II is a protein that plays a crucial role in cell growth and development. This antibody can be used in various immunoassay techniques, such as Western blotting and ELISA, to measure IGF-II levels in research applications.
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4 protocols using igf 2 antibody
1
Immunohistochemical Scoring of IGF-II in Prostate Tissue
2
Prostate Tissue Immunohistochemical Staining
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Using a microtome (Leica Biosystems, Nussloch, Germany), 8 µm thick slices were taken from formalin xed para n embedded (FFPE) prostate tissue blocks and mounted on Tomo microscope slides (Matsunami, Bellingham, Washington, USA). Slides were stained for IGF-II peptide using an IGF-II antibody (AbCam, Cambridge, UK) at a dilution of 1:600, on a Ventana BenchMark ULTRA™ machine (Roche, Oro Valley, Arizona, USA). Slides were scored rst by a pathologist and second, in-house, using a modi ed version of the Allred system, combining the proportion of tissue stained (a scale of 1-5) with the stain intensity (a scale of 1-3) to give a score out of 8 [35] . Currently, there is no standardised method of scoring prostate tissue.
3
Immunohistochemical Localization of IGF2 in Human Testis
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To examine the location of insulin-like growth factor II (IGF2) in human testicular tissues, we performed immunohistochemistry staining to detect the IGF2 expression. Tissues were cut into sections for immunoperoxidase staining after being treated with 4% PFA and paraffin wax. After the specific treatment with standard-procedure immunohistochemistry staining as described as Lian et al. [10 (link)], sections were incubated with IGF2 antibody (Abcam) overnight at 4°C and biotinylated secondary antibody (Abcam) for 2 h at room temperature.
4
Quantification and Analysis of Cellular Proteins
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Total proteins extracted from cultured cells were quantified by BCA assay, separated on SDS/polyacrylamide gels and transferred to a nitrocellulose filter. Anti-IGF1R, anti-IR, anti-Cyclin E1 (Cat# 4129, Cell Signaling Technology, Danvers, MA), anti-Caspase-3 (Cat# 9662, Cell Signaling Technology, Danvers, MA), phospho and total p44/p42 MAPK ERK1/2 (Cat# MAB-91982 and Cat# AB-82379, respectively, Immunological Sciences, Rome, Italy), total AKT (Cat# MAB-94324, Immunological Sciences, Rome, Italy), were used at 1:1000. Phospho-AKT (Ser473) antibody was used at 1:2000 (Cat# ABP-0637, Immunological Sciences, Rome, Italy) and IGF2 antibody (Cat# ab9574, Abcam, Cambridge, UK) was used 0.2 μg/ml. The detection of phosphorylated proteins was normalized on total proteins. GAPDH was used as housekeeping. Chemiluminescence was detected using the Chemidoc-IT Imaging System (UVP, Upland, CA) and densitometrical analysis was performed with NIH ImageJ software. Experiments were repeated at least 3 times.
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