Sample sections (5μm) were deparaffinized and rehydrated before commencing the immunofluorescence assay. Slides were fixed with 4% paraformaldehyde and blocked for nonspecific binding using 10% goat serum and 5% FBS in PBS (Millipore) for 2 hours at room temperature. NDRG1 (ab124689, Abcam) or PKCδ (ab182126, Abcam) primary antibodies were incubated at 1:100 dilutions overnight at 4°C. A secondary antibody coupled to Alexa Fluor™ 488 or Cy3 was added to each slide for 1 hour at room temperature. Samples were mounted using a DAPI solution containing ProLong Gold antifade and mounting medium (Invitrogen). Fluorescence images were taken by an Olympus BX51 microscope equipped with a 40× lens objective (Olympus America Inc.). Images were captured with an Olympus DP-6 digital camera and processed with TissueFAXS Viewer.
Ab182126
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab182126 is a monoclonal antibody product from Abcam. This antibody is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab124689, by Abcam (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Prolong gold antifade and mounting medium, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Bcl xl, by Abmart (1 mentions)
Vimentin, by Abmart (1 mentions)
Ab208035, by Abcam (1 mentions)
Ab76155, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Ab170099, by Abcam (1 mentions)
Gapdh, by Huabio (1 mentions)
Ab32445, by Abcam (1 mentions)
Ab133485, by Abcam (1 mentions)
Phospho erk 9101, by Cell Signaling Technology (1 mentions)
Phorbol 12 myristate 13 acetate tpa, by Merck Group (1 mentions)
Erk 4695, by Cell Signaling Technology (1 mentions)
Irdye 680cw, by LI COR (1 mentions)
Irdye 800rd, by LI COR (1 mentions)
9 protocols using ab182126
1
Immunofluorescence Assay for NDRG1 and PKCδ
2
Comprehensive Immunohistochemical Analysis
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We performed IHC analysis according to the staining procedure. The antibodies used are as follows: PRKCD (ab182126; Abcam, dilution 1:2000), Vimentin (Abmart, T55134, dilution 1: 200), ZO1 (Abmart, TA5145, dilution 1: 300), Bad (Abmart, T40052, dilution 1: 300), Bcl-xl (Abmart, T40057, dilution 1: 300), Ki67 (Servicebio, GB121141, dilution 1: 600).
3
Penile Tissue Protein Extraction and Analysis
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Total protein samples were extracted from penile tissues. The penile tissues were homogenized by precooled tissue lysates and centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatants were obtained. Protein concentrations were determined using a BCA protein assay kit (Solarbio, Beijing, China). Protein lysate of equal concentration was loaded on 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 30 min and 120 V for 60 min, and then electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad, CA, U.S.A.). The membrane was blocked for 1 h at room temperature with 5% skimmed milk powder. Then, membranes were incubated with primary antibodies against CaSR (1:1000; ab18200, Abcam), PLC (1:5000; ab76155, Abcam), PKCδ (1:5000; ab182126, Abcam), JNK (1:2000; ab208035, Abcam), p38 (1:1000, ab170099, Abcam), Bax (1:2000, ab32503, Abcam), GAPDH (1:5000, 60004-1-1g, HuaBio, Hangzhou, China) at 4°C overnight, followed the membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Finally, the protein bands were visualized using an enhanced chemiluminescence detection system (Clinx Science Instruments, U.S.A.).
4
Immunoblotting for Kinase Signaling
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Western blotting was followed, was described elsewhere [12 (link)]. Polyclonal rabbit antibody for Fli-1 (ab133485), PKCδ (ab182126), Phospho- PKCδ (ab133456), MEK (ab178876), Phospho-MEK (ab96379), BAD (ab32445), and Phospho-BAD (ab129192) were obtained from Abcam (Abcam, Cambridge, UK); ERK (#4695) and phospho-ERK (#9101) from Cell Signalling Technology (CST, Danvers, MA01923, USA), β-actin (20536–1-AP) and GAPDH (13937–1-AP), from Proto-Technology (Protein-Tech, Bucuresti, Romania). Antibody dilution according to the manufacturer instructions.
The inhibitor of MEK (U0126) [#S1102] were obtained from Sellectchem (Sellectchem, Houston, USA) and PKC agonist Phorbol 12-myristate 13-acetate (TPA) from Sigma, (Sigma, St. Louis, MO, USA). These compounds were dissolved in DMSO and added to the cells at indicated concentration, as described in the results section.
The inhibitor of MEK (U0126) [#S1102] were obtained from Sellectchem (Sellectchem, Houston, USA) and PKC agonist Phorbol 12-myristate 13-acetate (TPA) from Sigma, (Sigma, St. Louis, MO, USA). These compounds were dissolved in DMSO and added to the cells at indicated concentration, as described in the results section.
5
Immunoprecipitation and Immunoblotting of PKC Isoforms
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Freshly isolated or cultured vessel segments were prepared for immunoprecipitation, one‐dimensional protein gel electrophoresis and immunoblotting as described previously (Shi et al. 2017 a). The primary antibodies used were: mouse anti‐PKCε (dilution 1:500; sc‐1681; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti‐PKCθ (dilution 1:500; sc‐1680; Santa Cruz Biotechnology), mouse anti‐PKCη (dilution 1:500; sc‐136 036; Santa Cruz Biotechnology) and rabbit anti‐PKCδ (dilution 1:1000; ab182126; Abcam, Cambridge, MA, USA). Rabbit anti‐TRPC1 antibody (1 μg mL−1) was generated by GenScript (Piscataway, NJ, USA) using peptide sequences from a previously characterized putative extracellular region (Xu & Beech, 2001 ). Visualization were performed using anti‐rabbit and anti‐mouse secondary antibodies conjugated to IRDye 800RD or IRDye 680CW (dilution 1:10 000; Li‐Cor Biosciences, Cambridge, UK) as appropriate and the Odyssey Infrared Imaging System (Li‐Cor Biosciences). Protein band intensities were measured using Image Studio software (Li‐Cor Biosciences) and normalized to smooth muscle actin (dilution 1:2000; #ab5694; Abcam) when quantified.
6
Immunohistochemical Analysis of Pancreatic Tissue
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Pancreatic tissue sections (4 μm thick) were deparaffinized in xylene for 15 minutes, rehydrated using ethanol gradients and antigen retrieved by microwaving at 95°C for 5 minutes in 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by immersing the slides in peroxidase blocking buffer (3% hydrogen peroxide) for 10 minutes at room temperature. Further, slides were incubated with blocking buffer (5% BSA) for 1 hour at room temperature. Primary antibodies for PKC-δ (1:100, ab182126; Abcam, Cambridge, UK) and NF-κB p65 (1:100, sc-372; Santa Cruz Biotechnology) were then added and incubated at room temperature for 2 hours. Slides were rinsed in TBS and then, incubated with secondary antibody (k4003, polymer HRP-labelled anti rabbit; DAKO, Glostrup, Denmark) for 20 minutes at room temperature. Slides were again washed in TBS and visualized by incubating with diaminobenzidine substrate for 3 minutes at room temperature. Slides were then washed in distilled water and nuclei was counterstained using Mayer’s hematoxylin for 1 minute followed by two rinses in distilled water. Further, slides were dehydrated by serial immersion for 1 minute each in 70% ethanol and 95% ethanol followed by 2 minutes each in 100% ethanol and two changes of xylene. Finally, slides were mounted using Pertex mounting medium and coverslipped.
7
Monosynaptic Rabies Tracing in Cre Mice
8
PKCδ Immunohistochemistry Scoring Protocol
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Immunohistochemistry was carried out by Shanghai Runnerbio Biotechnology Co., Ltd. (Shanghai, China). Tissue Array were stained with primary antibodies against PKCδ (1:100, ab182126, Abcam). Scoring criteria of IHC: all sections were read by three pathologists under an ordinary light microscope and scored according to the German semi-quantitative scoring system. The specific scoring criteria is as follows: (1) staining intensity: 0: no staining; 1: mild staining; 2: moderate staining; 3: strong staining; (2) dying area: 0: < 10%; 1: 10%-25%; 2: 26%-50%; 3: 51%-75%; 4: > 76%. The IHC score of objective protein equals (1) × (2).
9
Signaling Pathway Inhibition Assay
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Peprotech (Rocky Hill, NJ, USA) supplied us with recombinant rat IL-1β. PKC-δ inhibitor rottlerin was obtained from MedChemExpress (Monmouth Junction, NJ). Abcam (Cambridge, MA, USA) offers PKC-δ (ab182126), JNK (ab124956), and p38 (ab32142) antibodies. The Cell Signaling Technology (Beverly, MA, USA) provided antibodies against p-PKC-δ (2055S) and p-p38 (4511S). Proteintech (ProteinTech Group, Chicago, IL, USA) provided antibodies against p-JNK (80024-1-RR), BAX(50599-2-Ig), caspase-3(66470-2-IG), and GAPDH (10494-1-AP).
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