Ovarian cortical tissue was sectioned in 5 μm slides and mounted on glass slides. Dehydration and antigen retrieval was performed as described elsewhere (Stubbs et al., 2005 (link)) followed by serum block (30 min), then primary antibody; anti-IGF2 rabbit polyclonal antibody (ab9574, Abcam, Cambridge, U.K.), (5 μg/ml) overnight at 4°C. This antibody was previously used and validated (Huang et al., 2010 (link)) and several other applications (ab9574-references.html">http://www.abcam.com/igf2-antibody-ab9574-references.html ). The sections were subsequently incubated in a 1:250 dilution of appropriate secondary antibody (donkey-anti-rabbit for IGF2) conjugated with Alexa Fluor 488 Dye (Life Technologies, Carlsbad, CA, U.S.A.). Sections were incubated in 1/3,500 Hoechst (Life Technologies, Carlsbad, CA, U.S.A.) followed by mounting with Dako Fluorescent Mounting Medium (Agilent Technologies, Santa Clara, CA, U.S.A) and analyzed using a LSM510 laser-scanning confocal microscope using a 63x C-Apochromat water immersion objective NA 1.2 (Carl Zeiss, Göttingen, Germany). Zen 2011 software (Carl Zeiss, Göttingen, Germany) was used for analysis and image capturing. The quantification of IGF2 immunofluorescence was done by as ImageJ (Jensen, 2013 (link)).
Ab9574
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab9574 is a laboratory equipment product manufactured by Abcam. It is a general-purpose item used in various scientific applications. The core function of this product is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Ab8227, by Abcam (3 mentions)
Ab121239, by Abcam (2 mentions)
Ab32572, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Imaging system, by Bio-Rad (2 mentions)
Ab3771, by Abcam (2 mentions)
Ecl kit, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Quantigene viewrna ish tissue assay kit, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Vector Laboratories (1 mentions)
Envision system hrp, by Agilent Technologies (1 mentions)
Tsa plus dig kit, by PerkinElmer (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Anti β tubulin antibody, by Merck Group (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Las 3000 imaging system, by Fujifilm (1 mentions)
Envision detection kit, by Agilent Technologies (1 mentions)
Ab6002, by Abcam (1 mentions)
21 protocols using ab9574
1
Quantitative Immunofluorescence Analysis of IGF2 in Ovarian Tissue
2
Western Blot Analysis of Cellular Proteins
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Total proteins were isolated from cultured cells using RIPA Lysis Buffer (Beyotime, Beijing, China) according to the manufacturer’s instructions and quantified using the BCA method (P0012S, Beyotime, Beijing, China). Approximately equal amounts of proteins in 20–40 μL were separated by 12% SDS-PAGE and transferred onto PVDF membranes. After blocked with 5% nonfat milk, the membranes were incubated first with primary antibodies against Nox4 (Ab9574, 1:1000, Abcam), NF-κB p65 (Ab3771,1:1000, Abcam), HIF-1α (Ab9574, 1:1000, Abcam), and β-actin (Ab8227, 1:1000, Abcam, Cambridge, MA) at 4°C overnight and then with appropriate HRP-conjugated secondary antibodies at room temperature for another 1 h. Positive signals were detected using an ECL kit (Millipore, Germany), and the bands were visualized using a Bio-Rad imaging system and quantitatively analyzed using Image J software.
3
Quantifying Protein Expression in Cultured Cells
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Total protein of cultured cells was isolated by RIPA Lysis Buffer (Beyotime, Beijing, China) according to the manufacturer's instructions. Approximately equal amounts of protein were separated by 12% SDS-PAGE and transferred into PVDF membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against Nox4 (Ab9574, 1:1000, abcam), NF-κB p65 (Ab3771,1:1000, abcam), HIF-1α (Ab9574, 1:1000, abcam) and β-actin (Ab8227, 1:1000, abcam, Cambridge, MA) at 4°C overnight, followed by incubation with appropriate HRP-conjugated secondary antibodies at room temperature for another 1 h. Positive signals were detected by using an ECL kit (Millipore, Germany), and bands were visualized under a Bio-Rad imaging system.
4
Immunohistochemical Analysis of CRC Proteins
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IHC for the target proteins was performed on formalin-fixed, paraffin-embedded tissue sections. The degree of positivity was initially classified according to scoring both the proportion of positive staining tumor cells and the staining intensities as previously described.39 (link) For histological scoring, three high-power fields were randomly selected from CRC and normal tissues. The primary antibodies used were anti-PLAGL2 (ab121239, Abcam), anti-IGF2 (ab9574, Abcam), and anti-β-catenin (ab32572, Abcam).
5
Immunohistochemical Analysis of Liver Markers
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The livers were fixed in phosphate-buffered 10% formalin for 24 h, and paraffin sections were then prepared. Immunohistochemical staining was carried out with the EnVision/HRP system (Dako, Carpinteria, CA, USA) on deparaffinized sections treated with Target Retrieval Solution (Dako). The antibodies used were as follows: anti-insulin-like growth factor 2 (IGF2) (ab9574; Abcam, Cambridge, UK), anti-α-fetoprotein (AFP) (14550-1-AP, for mouse tissues; Proteintech Group, Chicago, IL, USA), anti-AFP (A0008, for human tissues; Dako), and anti-trefoil factor 3 (TFF3) (Abbiotec, San Diego, CA, USA). 3,3′-Diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) was used for signal detection. For the detection of the TFF3 peptide, we applied signal amplification using the TSA Plus DIG Kit (PerkinElmer, Waltham, MA, USA). In situ hybridization for non-coding H19 mRNA was carried out on deparaffinized sections using the mouse H19 QuantiGene ViewRNA Probe Set (VB6-16706; Affymetrix, Santa Clara, CA, USA) and the QuantiGene ViewRNA ISH Tissue Assay Kit (Affymetrix).
6
Quantifying IGF2 and DNMT1 Levels
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Total protein was extracted using Mammalian Protein Extraction Reagent (M-PER; Thermo Scientific). For each sample, 10 µg of protein was resolved by SDS-PAGE (10%) and transferred to nitrocellulose membranes. The membranes were incubated overnight at 4 °C with anti-IGF2 (Abcam #ab9574), which enabled the detection of IGF2 precursor (approximate molecular weight: 20 kDa), and anti-DNMT1 (Santa Cruz Biotechnology #sc-10219) primary antibodies at a dilution of 1/500. A 1/1500 dilution of anti-β-Tubulin antibody (Sigma #T4026) was used as a loading control. The antigen–antibody complexes were visualized via chemiluminescence using ImmunoStar LD detection reagent from Wako (#290-69904) and scanned with a Fujifilm LAS-3000 imaging system.
7
Immunohistochemical Analysis of IGF2 in Breast Cancer
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To study the protein expression of IGF2, immunohistochemistry staining was performed on tissue sections of breast cancer using a TechMate TM50 autostainer (Dako, Glostrup, Denmark), a primary antibody against IGF2 (ab9574, Abcam, antigen retrieval pH6, incubation 1 h) and the EnVision Detection kit (Dako). Sections were counterstained with hematoxylin, dehydrated with ethanol and permanently coverslipped, as previously described [19 (link)].
8
Cartilage Degeneration and Synovitis Assessment
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Ten sections of knee joints spanning 200 μm were Safranin-O stained. Articular cartilage destruction was evaluated, according to the guideline of Osteoarthritis Research Society International [51 (link)]. Synovitis was graded using a 0–3 scoring system, with 0 = normal, 1 = moderate, and 3 = severe degeneration. Immunoreactivity was examined using Utx (ab253183, Abcam), H3K27me3 (ab6002; Abcam), Tfam (SC166965; Santa Cruz), Igf2 (ab9574; Abcam), Ezh2 (ab283273, Abcam), Eed (ab264566, Abcam), Suz12 (ab126577, Abcam), and Super Sensitive™ IHC Detection Systems (BioGenex Laboratories). Immuno-labeled chondrocytes within each field (50 μm2) were counted (nine fields in three sections for each specimen).
9
Immunohistochemical Analysis of IGF2 Expression
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The protein level of IGF2 in tissues was examined by immunohistochemistry (IHC). Briefly, IHC was carried out on paraffin-embedded tissue sections from HB tissues and matched normal tissues (n=27). After deparaffinization, hydration, antigen retrieval and blocking of endogenous peroxidase and nonspecific binding, tissue sections were incubated with primary antibody against IGF2 (abcam, ab9574, Cambridge, UK) overnight at 4°C, followed by incubation with biotinylated secondary anti-Rabbit IgG antibody and peroxidase-labeled streptavidin at room temperature for 15 min. Then, the antigen was visualized after incubation with diaminobenzidine chromogenic substrate at room temperature for 10 min. Representative images were photographed under an inverted microscope (Leica). Two specialized pathologists who were blinded to the clinical information of these tissue specimens were invited to analyze the staining intensities of tissues at two magnifications (100× and 200×).
10
Investigating IGF2 Localization in sh_circHMGCS1 HepG2 Cells
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HepG2 cells infected with sh_circHMGCS1 or sh_NC were seeded into 24-well plates with coverslips. The next day, cells were fixed and permeabilization. After blocking with 2% BSA, cells were incubated with the antibody against IGF2 (Abcam, ab9574), followed by incubation with the secondary antibody (Cell Signaling Technology, Boston, Massachusetts, USA). Afterwards, cells were stained with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Finally, the slides were analyzed using a confocal fluorescence microscope (Zeiss, Germany).
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