Tyrosine phosphorylation of synaptophysin has been shown to be necessary for long-term potentiation, a cellular basis of learning and memory [26 (link)]. Rat hippocampal was dissected after snap frozen. Western blots were performed and analyzed as previously described [6 (link)]. Antibodies used for Western blots were as follows: synaptophysin (Abcam, Cambridge, MA, USA) and rabbit anti-PSD95 antibody (Abcam), and β-actin (Cell Signaling Technology).
Rabbit anti psd95 antibody
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti-PSD95 antibody is a primary antibody used for the detection and analysis of PSD95 (Postsynaptic Density Protein 95) in various biological samples. PSD95 is a key scaffolding protein found in the postsynaptic density of excitatory synapses.
Automatically generated - may contain errors
Lab products found in correlation
Synaptophysin, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
G actin f actin in vivo assay kit, by Cytoskeleton (1 mentions)
Fd rapid golgistain kit, by FD NeuroTechnologies (1 mentions)
Rabbit anti gapdh antibody, by Affinity Biosciences (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Enhanced chemiluminescence detection system, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti bdnf antibody, by Alomone (1 mentions)
Rabbit anti trkb antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti synaptophysin, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mowiol, by Merck Group (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Goat anti gfp antibody, by Rockland Immunochemicals (1 mentions)
Fluoview version 3, by Olympus (1 mentions)
Mouse anti nf200 antibody, by Merck Group (1 mentions)
Goat anti cgrp antibody, by Abcam (1 mentions)
Rabbit anti rfp antibody, by Abcam (1 mentions)
5 protocols using rabbit anti psd95 antibody
1
Synaptophysin Tyrosine Phosphorylation and LTP
2
Molecular Profiling of Synaptic Proteins
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A mouse anti-srGAP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A rabbit anti-PSD95 antibody, a rabbit anti-NMDAR2B (phospho Y1472) antibody and a mouse anti-NMDAR2B antibody were obtained from Abcam (Cambridge Science Park, Cambridge, UK). A mouse anti-active Rac1 antibody and the Rac1 Activity Assay Kit were obtained from New East (New East Biotechonology Co. Ltd, Wuhan, China). A rabbit anti-GAPDH antibody was purchased from Affinity Biosciences (Cincinnati, OH, USA). The rac1 activator CN04 was obtained from Cytoskeleton Inc. (Cat. CN04-A, Cytoskeleton, Colorado, USA). The FD Rapid GolgiStain Kit was obtained from FD Neurotechnologies, Inc (Guilford, MD, USA). The G-Actin/F-actin In Vivo Assay Kit was obtained from Cytoskeleton, Inc.
3
Western Blot Analysis of Synaptic Proteins
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According to a previously described method [13 (link),21 (link)], western blot was conducted. From the peri-infarct cortex and hippocampus, total protein was extracted using lysis buffer containing 150mM NaCl, 1mM phenylmethylsulfonyl fluoride, 1% Nonidet P40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 100-mg/mL leupeptin, 50mM Tris-HCl (pH, 7.5). Protein was quantified using a Bradford protein assay (Bio-Rad, Hercules, CA, USA). Protein, 30 µg, was separated on SDS polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Chicago, IL, USA). The membrane was blocked with skim milk, then membrane was incubated by mouse anti-β-actin antibody (1:1,000; Santa Cruz Biotechnology), rabbit anti-PSD-95 antibody (1:1,000; Abcam, Cambridge, UK), rabbit antisynaptophysin (1:1,000; Abcam), rabbit antiBDNF antibody (1:1,000; Alomone Labs, Jerusalem, Israel), and rabbit anti-TrkB antibody (Santa Cruz Biotechnology) at 4°C during overnight. After washing, species appropriate horseradish peroxidase-conjugated secondary antibodies were incubated for 1 hour. Band detection was performed using the enhanced chemiluminescence detection system (Santa Cruz Biotechnology).
4
Immunohistochemistry of Zebrafish Brain
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Following humane killing in 0.1% tricaine, zebrafish embryos were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), cryoprotected in 30% sucrose in PBS solution, embedded in OCT (Tissue-Tek) and cryostat sectioned at 12 µm thickness. Antibody staining was carried out at room temperature using standard protocols. Antigen retrieval was performed by incubating slides in boiled 10 mM sodium citrate (pH 6) until the solution was cooled down to room temperature. Slides were then blocked in 5% foetal bovine serum (FBS) for 30 min and incubated overnight in rabbit anti-PSD95 antibody (1:100, Abcam, cat. number ab18258) diluted in the same block solution. On the second day, slides were rinsed using PBS and then incubated for 2 h in anti-Rabbit Alexa Fluor 488 (1:500, Thermo Fisher Scientific, cat. number A11001) diluted in 5% FBS. Nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:10000, Sigma-Aldrich, cat. number D9542-10MG) in PBS solution. Sections were mounted in Mowiol (Sigma-Aldrich, cat. number 81381-250 G).
5
Immunohistochemical Profiling of Spinal Cord and DRG
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Mice were anesthetized with pentobarbital sodium and transcardially perfused with saline followed by 4% paraformaldehyde. Vibratome sections (30 μm) of the spinal cord and brain or Cyrostat-sections (16 μm) of the L4/L5 DRG were immunostained using standard protocol with goat anti-NR1 antibody (Santa Cruz, 1:100), biotinylated isolectin B4 antibody (vector laboratories, 1:200), rabbit anti-CGRP antibody (Calbiochem, 1:500), goat anti-CGRP antibody (Abcam, 1:500), mouse anti-NF200 antibody (Sigma-Aldrich, 1:200), rabbit anti-PSD-95 antibody (Abcam, 1:500), rabbit anti-synaptophysin antibody (Abcam, 1:500), goat anti-GFP antibody (Rockland, 1:500), rabbit anti-RFP antibody (Abcam, 1:500), mouse anti-Flag (Abbkine, 1:800). The number of immunoreactive neurons per DRG section was counted and numbers were averaged over 10 sections per mouse and 4 mice per treatment group. All images were captured with Olympus Fluoview version 3.1 software in an Olympus confocal microscope. See also the list of antibodies used in Supplementary Table 1 .
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