Spinal cord sections were prepared from intact and TBI mice. Mice were perfused transcardially with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Spinal cords were dissected, postfixed in the same fixative, immersed overnight in PBS containing 30% sucrose, and then embedded in Tissue-Tek OCT and frozen at −80 °C until use. Sections were prepared using a cryostat (20 µm thickness) and mounted on Matsunami adhesive-coated slides (Matsunami, Osaka, Japan). Cryostat sections were incubated with blocking solution containing 5% BSA and 0.1% Triton X-100 in PBS for 1 h at room temperature, followed by overnight incubation with primary antibodies (anti-PKCγ, Santa Cruz; anti-GFAP, Sigma-Aldrich; anti-Iba1, Wako, anti-NeuN, Millipore; anti-tdTomato, SICGEN; anti-GFP, Thermo Fischer Scientific; anti-HDAC2, Abcam; anti-HDAC1, Abcam; anti-Chx10, Exalpha biologicals; anti-Olig2, Immuno-Biological Laboratories) at 4 °C. Immunoreactivity was visualized using Alexa Flour 488- or 568-conjugated secondary antibodies (Thermo Fischer Scientific). Coverslips were then placed on the slides with mounting medium (Dako). Nuclei were stained using 4′, 6-diamidino-2-phenylindole (DAPI). Images were captured using a laser scanning confocal microscope (FV-1200, Olympus) or a fluorescence microscope (IX83, Olympus).
Anti pkcγ
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-PKCγ is a laboratory reagent used for the detection and quantification of the PKCγ protein in biological samples. It functions as a specific antibody that binds to the PKCγ protein, enabling its identification and measurement through various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti pkcδ, by Santa Cruz Biotechnology (2 mentions)
Anti pkcα, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Anti hdac1, by Abcam (1 mentions)
Anti hdac2, by Abcam (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Anti neun, by Merck Group (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Fv1200, by Olympus (1 mentions)
Anti caspase 3 and 9, by Cell Signaling Technology (1 mentions)
Anti pkcζ, by Santa Cruz Biotechnology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Anti epcam, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti atg5, by Cell Signaling Technology (1 mentions)
Anti lc3a b, by Cell Signaling Technology (1 mentions)
Sc 208, by Santa Cruz Biotechnology (1 mentions)
8 protocols using anti pkcγ
1
Spinal Cord Cryosectioning and Immunostaining
2
Protein Kinase C Isoform Analysis
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After treatment with desired concentrations of Bis-I, Gö6976, and Rottlerin, cells were harvested and washed in PBS. The extracts were prepared by resuspending cell pellets in lysis buffer (50mM Tris-HCI, pH 8.0, 150 mM NaCI, 1% Triton X-100, 5 mM EDTA), briefly sonicated and centrifuged at 15000xg for 15 min at 4°C. The protein concentrations were determined using BCA assay (Thermo). SDS-PAGE and immunoblotting were performed. Nitrocellulose membranes were probed with anti-PKCα, anti-PKCδ, anti-PKCη, anti-PKCγ, anti-PKCζ, anti-GAPDH (sc-208, sc-213, sc-215, sc-211, sc366126, sc-25778, Santa Cruz Biotechnology), anti-PKCβ, anti-PKCϵ, anti-claudin-1, −3, −4, and −7, anti-CD9, anti-CD82, Tspan-8 (Abcam), anti-caspase 3 and 9, anti-LC3A/B, Anti-Atg-5, anti-EpCAM, anti-E-cadherin, anti-vimentin (Cell Signaling). Immunoblotting signals were developed using chemiluminescence. All experiments were performed in triplicate.
3
Western Blotting of Platelet Proteins
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Western blotting was performed by modifying a previously described procedure [26 (link)]. Briefly, platelets were lysed in buffer A and B and centrifuged at 20,000× g for 30 min, and the supernatant was collected to obtain the cell membrane lysate. Then, proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and reacted with anti-PKC-α antibody (1:500, Santa Cruz), anti-PKC-βI (1:500, Santa Cruz), anti-PKC-βII (1:500, Santa Cruz), anti-PKC-γ (1:500, Santa Cruz), anti-PKC-δ (1:500, Santa Cruz), or anti-actin (1:3000, Cell Signaling) overnight. All samples were analyzed using a LAS 4000 mini (Fuji Photo Film, Tokyo, Japan). This experiment was repeated a total of three times.
4
Quantitative Western Blotting and Co-IP
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For Western blotting, anti-phospho-PKCγ (Abcam) and anti–β-actin (loading control; Sigma-Aldrich) were used to detect IL-4 effects on synaptosomes. Anti-P44/42 MAPK (ERK1/2) and anti–phospho-P44/42 MAPK (ERK1/2) were used on Western blots of IL-4–stimulated dissociated neurons (50 ng/ml, 10 min). DyLight 800/600-coupled secondary antibodies were used for quantitative analysis of the proteins using the Li-Cor Odyssey FC imaging system (Li-Cor Bioscience) and ImageJ. For co-IP, performed as previously described (Vogelaar et al., 2018 (link)), synaptosome lysates were incubated with anti–IL-4α (BD Bioscience) on protein-A/G agarose beads overnight at 4°C in IP buffer (20 mM Tris-HCL pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 µg/ml pepstatin; Sigma-Aldrich). Precipitation was performed by centrifugation with descending speeds (6,000–3,000 g) for 2 min. Blots were incubated with anti–β-actin (Sigma-Aldrich), anti-PKCγ (Santa Cruz), and anti–GAP-43 (Novus Biologicals; Table 1 ). Raw data were extracted from the Li-Cor software and imported to ImageJ using the bio-formats plugin. Bands were outlined with the rectangular icon and intensities were plotted in curves. The area under the curve was calculated for each sample.
5
Clematichinenoside Signaling Pathway Study
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Clematichinenoside (95.3% purity) was provided by China Pharmaceutical University. PD98095, GF109203X, BI-D1870, and KG-501 were purchased from Shang Hai Haoran Biological Technology CO., LTD (Shanghai, China) Dulbecco's modified Eagle's medium (DMEM, high glucose) and newborn calf serum were products of Gibco. Anti-ERK1/2, Anti-phospho-ERK1/2, anti-PKCα, anti-PKCβI/βII, anti-PKCγ, anti-phospho-PKCα, anti-phospho-PKCβI/βII, anti-phospho-PKCγ, anti-p90RSK, anti-phospho-p90RSK, anti-CREB, anti-phospho-CREB, anti-bcl-2, anti-bcl-xl, and anti-bax were purchased from Santa Cruz Biotechnology, Inc. Primers for PCR were obtained from Shanghai Sangon Biotech Co., Ltd. RT-PCR kit was bought from Dalian Takara Biotechnology Co., Ltd.
6
Molecular Mechanisms in Cardiac Hypertrophy
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Protocatechuic acid (3,4-dihydroxybenzoic acid; cat no. 37580), isoproterenol (cat no. I5627), and actinomycin D (cat no. A9415) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Anti-GAPDH (cat no. sc-32233), anti-β-actin (cat no. sc-47778), anti-BNP (cat no. sc-271185), anti-ROCK1 (cat no. sc-17794), anti-PKCγ (cat no. sc-166385), and anti-Sp1 (cat no. sc-17824) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). Alexa Fluor 488 phalloidin (cat no. A12379) and Alexa Fluor 568 goat anti-mouse IgG (cat no. A11004) were purchased from Invitrogen (Eugene, OR, USA). Wheat germ agglutinin Alexa Fluor 488 conjugate (cat no. W11261) was purchased from ThermoFisher Scientific (Waltham, MA, USA). Human PKCγ cDNA clone expression plasmid was purchased from Sino Biological Inc. (cat no. HG112420ANG, Wayne, PA, USA).
7
Spinal Cord Tissue Immunostaining Protocol
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Male GlyT2-iCre-tdTomato mice (4–8 weeks) were anesthetized deeply by injecting pentobarbital sodium (50 mg/kg) intraperitoneally. The mice were immediately perfused in order with the saline and ice-cold paraformaldehyde (PFA). Then, the spinal cord tissue was extracted and post-fixed in the PFA (4%), followed by dehydrating with 20% and 30% sucrose for 24 h, respectively, at 4°C. Then, the spinal cord was cut into sections (25 μm) by Cryostat Microtome (Leica, Germany). After being washed in phosphate buffer saline (PBS), they were blocked in donkey serum (5%) for 2 h at room temperature (RT) and subsequently incubated in 2.5% donkey serum-containing primary antibodies: anti-Pax2 (rabbit, 1:500; Abcam, USA), anti-Glycine (rabbit, 1:200; Abcam, USA), anti-SP (guinea pig, 1:500; Abcam, USA), anti-CGRP (goat, 1:500; Abcam, USA), anti-PKCγ (rabbit, 1:400; Santa Cruz, USA) for 12–18 h at 4°C. After that, they were incubated with secondary antibodies which included donkey anti-rabbit IgG with Alexa Fluor 488 (1:500; Molecular Probes, USA), donkey anti-guinea pig IgG conjugated with Alexa Fluor 488 (1:500; Molecular Probes, USA), donkey anti-goat IgG with Alexa Fluor 488 (1:500; Molecular Probes, USA), respectively, for 3 h at RT. Besides, some of the fluorescent spinal cord slices were directly incubated with the IB4 (1:200; Vector Laboratories, USA).
8
Endothelial Cell Culture and Molecular Characterization
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Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC. HUVECs were cultured with M199 basal medium supplemented with low-serum growth supplement and penicillin (50 IU/ml)-streptomycin (50 μg/ml). trypsin-EDTA was used to passage cells. M199 and trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). Low-serum growth supplement was purchased from Cascade (Portland, OR, USA). Additionally. Baicalein, AICAR, Compound C, Apocynin, penicillin and streptomycin were all purchased from Sigma (St. Louis, MO, USA). Anti-β-actin, anti-AMPK, anti-AMPK-α, anti-phospho AKT, anti-LOX-1, anti-PKCα, anti-PKCβ, anti-PKCγ, anti-PKCδ, I-κB, NF-κBp65, anti-Cu, Zn SOD, anti-Mn SOD were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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