Retinas were isolated in cold oxygenated mouse artificial cerebrospinal fluid (mACSF, pH 7.4, 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1 mM NaH2PO4, 11 mM glucose, and 20 mM HEPES). Retinas were flattened onto filter paper (Millipore, HABP013) and fixed for 15 mins in 4% (wt/vol) paraformaldehyde prepared in mACSF. Retinas were rinsed in phosphate buffer (PBS) and then incubated in a blocking solution (5% donkey serum and 0.5% Triton X-100). The retinas were next incubated with primary antibody over 3 nights at 4°C. Primary antibodies used were anti-PKC (1:1000, mouse, Sigma; RRID:AB_477375 ), anti-Dsred (rabbit 1:1000, Clontech), anti-synaptotagmin 2 (1:1000, mouse, Znp-1 Zebrafish International Resource center; RRID:AB_10013783 ), anti-calbindin antibody (rabbit, 1:1000, Swant Inc; RRID:AB_10000340 ), anti-GABAAβ2/3 (mouse, 1:500 MilliporeSigma) anti-GABAAα1 receptor subunit (polyclonal guinea-pig, 1:5000, kindly provided by J.M. Fritschy), and anti-GABACρ receptor subunit (1:500, rabbit, kindly provided by R. Enz, H. Wassle, and S. Haverkamp). Retinas were thereafter incubated in secondary antibody solution using anti-isotypic Alexa Fluor (1:1000, Invitrogen) conjugates. Retinas were finally mounted on slides with Vectashield antifade mounting medium (Vector Labs; RRID:AB_2336789 ).
Anti pkc
Manufactured by Merck Group
Sourced in China
Anti-PKC is a laboratory reagent that specifically inhibits the activity of the protein kinase C (PKC) enzyme. It is used as a tool in scientific research to study the role of PKC in cellular processes and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Habp013, by Merck Group (2 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (2 mentions)
Anti dsred, by Takara Bio (1 mentions)
Anti pka, by Abcam (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Anti sod1, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti fn, by Santa Cruz Biotechnology (1 mentions)
Anti rage, by Merck Group (1 mentions)
Anti inos, by Abcam (1 mentions)
Anti ctbp2, by BD (1 mentions)
Em201 electron microscope, by Philips (1 mentions)
4 protocols using anti pkc
1
Immunostaining of Mouse Retina Tissue
2
Glomerular Protein Expression Analysis
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Glomerular protein was extracted with SDS protein lysate (KeyGEN, Nanjing, China), separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. PVDF membrane was blocked with 5% skim milk containing 0.05% Tween-20 and incubated with individual primary antibodies, including anti-FN (Santa Cruz Biotechnology, Dallas, TX), anti-RAGE, anti-PKC (Sigma-Aldrich, Shanghai, China), anti-PKA (Abcam, UK), anti-iNOS (Abcam, UK), anti-SOD1 (Santa Cruz, USA) and anti-actin (Sigma-Aldrich, Shanghai, China). Then HRP-conjugate secondary antibody was utilized and color development achieved with enhanced chemiluminescence (ECL) reagent (EMD Millipore, Billerica, MA, USA).
3
Immunolabeling of Retinal Whole-Mounts
4
Immunohistochemical Localization of PKC
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The same fixation was used as described for light microscopy. Transverse frozen sections, 30-40 µm thick, were obtained on a freezing microtome and collected in phosphate buffer (PB) at room temperature, which is equivalent to freeze-thawing. Retinal sections were incubated for 72 h with diluted antisera (anti-PKC, 1:400, mouse monoclonal, Sigma).
After rinsing, the tissue was incubated in a Poly-HRP-goat anti-mouse IgG (PowerVision, ImmunoVision Technologies Co., Springdale, AR). To visualize the peroxidase, the material was incubated in a Tris-HCl diaminobenzidine (DAB, 0.05%) solution containing 0.03% H2O2. The DAB reaction product was subsequently intensified by the gold-substituted silver peroxidase method (Gorcs et al., 1986) .
Sections were rinsed in sodium cacodylate buffer 0.1 M (pH 7.4) and post-fixed for 20 min in 1% OsO4 supplemented with 1.5 % potassium ferricyanide in sodium cacodylate buffer. After rinsing in sodium cacodylate buffer, the material was dehydrated and embedded in epoxy resin. Ultrathin sections were observed in a Philips EM201 electron microscope (Philips, The Netherlands) and/or Technai (CM) 12 electron microscope (FEI, The Netherlands). Sections examined with the Philips electron microscope were counter-stained with uranyl acetate and lead citrate.
After rinsing, the tissue was incubated in a Poly-HRP-goat anti-mouse IgG (PowerVision, ImmunoVision Technologies Co., Springdale, AR). To visualize the peroxidase, the material was incubated in a Tris-HCl diaminobenzidine (DAB, 0.05%) solution containing 0.03% H2O2. The DAB reaction product was subsequently intensified by the gold-substituted silver peroxidase method (Gorcs et al., 1986) .
Sections were rinsed in sodium cacodylate buffer 0.1 M (pH 7.4) and post-fixed for 20 min in 1% OsO4 supplemented with 1.5 % potassium ferricyanide in sodium cacodylate buffer. After rinsing in sodium cacodylate buffer, the material was dehydrated and embedded in epoxy resin. Ultrathin sections were observed in a Philips EM201 electron microscope (Philips, The Netherlands) and/or Technai (CM) 12 electron microscope (FEI, The Netherlands). Sections examined with the Philips electron microscope were counter-stained with uranyl acetate and lead citrate.
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