The following antibodies were used: βIII-tubulin (Santa Cruz Biotechnology sc-58888, clone TUJ-1), GAP43 (Millipore AB-5220), Tau (generous gift of Dr Lorenzo-Benayas, University of Extremadura), GAPDH (Cell Signaling 2118, clone 14C10), OCT4 (Santa Cruz Biotechnology sc-5279, clone C-10), NANOG (AbCam Ab-21624), AGO2 (Millipore 03–110), AHR (ENZO Life Sciences BML-SA210 and Immunostep 130605–1) and β-Actin (Sigma A2066).
Gap43
Manufactured by Merck Group
Sourced in United States
GAP43 is a laboratory reagent used in biochemical and cell biology research. It is a protein that plays a key role in the growth and development of neuronal axons. GAP43 is commonly used as a marker for neuronal plasticity and regeneration in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
β1 ar, by Bioworld Technology (2 mentions)
Nerve growth factor (ngf), by Signalway Antibody (2 mentions)
β2 ar, by Signalway Antibody (2 mentions)
Gm130, by BD (2 mentions)
P75ntr, by Merck Group (2 mentions)
Gapdh, by Merck Group (2 mentions)
Goat anti chicken 488, by Thermo Fisher Scientific (2 mentions)
Dpx mounting media, by Electron Microscopy Sciences (2 mentions)
βiii tubulin, by Santa Cruz Biotechnology (1 mentions)
Nanog, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
β3 ar, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Tapestation 2100, by Agilent Technologies (1 mentions)
Hrp conjugated, by Thermo Fisher Scientific (1 mentions)
Alexa fluor conjugated secondary antibody, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
20 protocols using gap43
1
Antibody Panel for Stem Cell Research
2
Protein Expression Analysis of Myocardial Infarction
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The tissues from the LV infarct border area (5 mm) was homogenized. Total proteins were extracted with RIPA lysis buffer containing protease inhibitors (Roche, Indianapolis, IN, USA). Protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membrane was incubated with the following diluted primary antibodies: TH (1∶500, Millipore), GAP43 (1∶1000, Millipore), NGF (1∶500, Signalway Antibody), β1-AR (1∶2000, Bioworld), β2-AR (1∶1000, Signalway Antibody), β3-AR (1∶800, Santa Cruz), NOS2 (1∶500, Cell Signaling, Beverly, MA, USA), phosphorylation of NOS2 at serine residue 1177 (p-NOS2ser1177, 1∶500, Cell Signaling), NOS1 (1∶400, Signalway Antibody), at 4°C overnight. Following washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1∶10000 dilution, Jackson, ImmunoResearch, USA). GAPDH was used as an internal control. Protein bands were subsequently detected with enhancedchemiluminescence and sections were exposed to X-ray film.
3
GCF Membrane Sealing Assay
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Membrane sealing and thus protection of GC-specific molecular contents was assessed using either an RNase or protease protection assay, as described in detail elsewhere50 (link). In brief, isolated GCF samples were treated in parallel with either nothing (control; ctrl), 0.1% TritonX-100 detergent (D-ctrl), RNAse/protease only (R/P-ctrl), or a combination of 0.1% TritonX-100 and either RNase or protease (D+R/P) and incubated for 2 hours at 4°C. For RNase protection assays, samples were processed for RNA extraction, and quality of extracted RNA was assessed using an Agilent Tapestation 2100 with a High Sensitivity RNA kit, using the manufacturer’s standard protocol. For protease protection assays, samples were processed for western blotting as described above, and probed with primary antibodies against the subcellular markers GAP43 (#MAB347 Millipore), Actb (#A5441 Sigma), Gm130 (#610823 BD Biosciences), and MAP2 (#M1406 Sigma), followed by secondary antibodies that were isotype-specific, HRP-conjugated, and cross-absorbed (Life Technologies).
4
Cardiac Nerve Regeneration Imaging
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Immunofluorescence stainings were performed in cardiac tissue to identify catecholaminergic phenotype (by colocalization of TH-immunoreactivity (ir) and PGP9.5-ir) and sympathetic re-innervation (by TH-ir and growth associated protein 43 (GAP43)-ir) in cardiac nerve bundles. Slides were deparaffinized and antigen retrieval performed as described above. Tissue was blocked with 5% donkey serum and 2% BSA solution and incubated in primary antibodies GAP43 (1∶100; Millipore; Billerica, MA), PGP9.5 (1∶100), and TH (1∶200) for 2 days in 4°C. The sections were then incubated with alexafluor-conjugated secondary antibody (1∶1000) against the appropriate species and coverslipped with mounting media with DAPI (Vector Laboratories, Burlingame, CA). Brain tissue from a rhesus fetus at embryonic day 38 was used as a positive control for GAP43.
5
Quantitative Analysis of Cardiac Sympathetic Nerves
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Briefly, the paraffin sections were incubated with the following diluted primary antibodies overnight at 4°C: TH (1∶400, Millipore, Billerica, MA, USA), GAP43 (1∶500, Millipore), NGF (1∶100, Signalway Antibody, Pearland, TX, USA). β1-AR (1∶150, Bioworld, Atlanta, Georgia, USA), and β2-AR (1∶150, Signalway Antibody). Following this incubation, sections were exposed to a secondary antibody for 30 min at room temperature. The stains were developed using diaminobenzidine as a chromogen. The sections were then counterstained with hematoxylin and examined by conventional light microscopy (Olympus BX51, Olympus Optical, Tokyo, Japan). Six sections from each group were scanned, with 10 fields per section viewed, and the value of mean optical density (OD) was calculated by IPP 6.0. The density of stained cardiac sympathetic nerves was determined using IPP 6.0 and expressed as the nerve area divided by the total area examined (um2/mm2). The nerve density of each slide was determined by the mean density of nerves calculated from all three selected fields.
6
Evaluating Synaptic Plasticity in Ischemic Stroke
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Rats were deeply anesthetized and perfused with 0.9% saline followed by 4% paraformaldehyde at 7 d after MCAO. Brains were taken out and immersed in sucrose for dehydration (sucrose of 15 and 30% successively). Coronal sections were cut at 10-μm thickness by freezing microtome (Leica, Germany). After being permeabilized with 0.3% Triton X-100 and blocked by goat serum working liquid, sections were incubated at 4°C overnight with primary antibodies: synaptophysin (SYN, 1:200, Millipore, USA) and growth-associated protein-43 (GAP-43, 1:250, Millipore, USA). The next day after being washed in Tris-buffered saline (TBS), sections were incubated with goat anti-mouse/rabbit IgG H&L secondary antibody (1:200, Abcam, USA) for 2 h at room temperature and mounted using anti-fade solution with DAPI (Solarbio, Shanghai, China). The stained sections were captured under a fluorescent laser scanning confocal microscope (ZEISS, Germany) and analyzed with Image-Pro Plus 6.0.
7
Antibody Characterization for Immunohistochemistry and Western Blotting
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P-STAT3-Ser727 and P-STAT3-Tyr705 antibodies, both from Cell Signaling Technology, were used at 1:50 for immunohistochemistry and 1:2000 for Western blotting. Other antibodies for Western blotting were Cyclin D1 (1:200; Santa Cruz Biotechnology), N-Cadh (1:500; BD Transduction Laboratories), p75NTR (1:1000; Millipore), GAP43 (1:500; Millipore), c-Jun (1:1000; Cell Signaling Technology), and GAPDH (1:5000; Sigma-Aldrich). HRP-conjugated secondary antibodies (1:2000 in blocking solution) were from Cell Signaling Technology. For immunohistochemistry, incubation with MBP antibodies (1:10,000; Covance) or 324 rat anti-mouse Ig L1 antibodies (1:10) were followed by anti-mouse Ig AlexaFluor-488 (1:500; Invitrogen) or anti-rat Ig AlexaFluor-488 (1:500; Invitrogen), respectively. Incubation with antibodies to Ki67 (1:100; Abcam) and SOX10 (1:100; R&D Systems) were followed by biotinylated anti-rabbit IgG (1:600; GE Healthcare) and anti-goat Ig AlexaFluor-488 (1:1000; Invitrogen) antibodies, respectively. The Ki67 sections were then incubated with AlexaFluor-488 streptavidin (1:500; Invitrogen). Caspase-3 antibody (1:100; Cell Signaling Technology) was followed by anti-rabbit Ig Cy3. S100 antibody (1:1000) was from Dako.
8
Standardized Western Blot Technique
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Western blots were performed using standard tris-glycine SDS-PAGE protocols. Samples had normalized protein concentration assessed using a Bradford assay (#23246, Thermo). Resolved proteins were electroblotted onto PVDF membranes using semi-dry transfer, blocked for 30 min in TBS supplemented with 0.1% Tween-20 and 5% skim milk, and incubated with primary antibodies (GAP43 #MAB347 Millipore; Actb #A5441 Sigma; Gm130 #610823 BD Biosciences; MAP2 #M1406 Sigma; WAVE1 #ab272916 abcam; WAVE2 #3659T Cell Signaling Technologies; WAVE3 #2806S Cell Signaling Technologies; Cpeb4 #ab224162 abcam; Rbms1 #ab150353 abcam; Tau #AF3494 RR&D) in TBS supplemented with 0.1% Tween-20 and 3% BSA over night at 4°C. The next day, the membrane was washed three times in TBS supplemented with 0.1% Tween-20, incubated for 2 hours at room temperature with secondary antibodies that were isotype-specific, HRP-conjugated, and cross-absorbed (Life Technologies), then washed again three times with TBS supplemented with 0.1% Tween-20. Immunoreactive bands were visualized through detection of chemiluminescence using a CCD camera imager (FluoroChemM, Protein Simple). Quantification of signal intensity in the various bands was performed using the standard Fiji software package.
9
Protein Detection in Ischemic Brain
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To detect levels of proteins involved in inflammation and regeneration after brain ischemia, mice stroke affected lateral portions of the hemisphere were isolated and snap-frozen in liquid nitrogen and stored at −80 °C. Proteins were isolated by mechanical homogenisation in SDS-urea-β-mercaptoethanol solution containing proteinase inhibitor on ice. Electrophoresis and transfer on the PVDF-membrane was done using BioRad Mini-PROTEAN3 System (BioRad, Hercules, CA, USA).
Primary antibodies were β3-Tubulin (5568 S, Cell Signalling Technology, Beverly, MA, USA) as a loading control, DLG4 (PSD95) (3450, Cell Signalling Technology Beverly, MA, USA), GAP43 (AB5220, Merck, Kenilworth, NJ, USA), synaptophysin clone SY38 (MAB5258, Merck, Kenilworth, NJ, USA), and anti-cleaved CASP3 (Cell Signaling, Danvers, MA, USA). Secondary antibodies were goat anti-mouse, and goat anti-rabbit (32430 and A27011, Thermo Fisher Scientific, Waltham, MA, USA). The chemiluminiscent signal achieved by Western Lightning – EXL Enhanced Chemiluminiscence Substrate (Perkin Elmer, Waltham, MA, USA) was imaged by ChemiDoc MP System (BioRad, Hercules, CA, USA) and analysed using Image Lab Software (BioRad, Hercules, CA, USA).
Primary antibodies were β3-Tubulin (5568 S, Cell Signalling Technology, Beverly, MA, USA) as a loading control, DLG4 (PSD95) (3450, Cell Signalling Technology Beverly, MA, USA), GAP43 (AB5220, Merck, Kenilworth, NJ, USA), synaptophysin clone SY38 (MAB5258, Merck, Kenilworth, NJ, USA), and anti-cleaved CASP3 (Cell Signaling, Danvers, MA, USA). Secondary antibodies were goat anti-mouse, and goat anti-rabbit (32430 and A27011, Thermo Fisher Scientific, Waltham, MA, USA). The chemiluminiscent signal achieved by Western Lightning – EXL Enhanced Chemiluminiscence Substrate (Perkin Elmer, Waltham, MA, USA) was imaged by ChemiDoc MP System (BioRad, Hercules, CA, USA) and analysed using Image Lab Software (BioRad, Hercules, CA, USA).
10
Immunohistochemical Analysis of Neural Markers
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Immunohistochemistry was performed on formalin‐fixed, paraffin‐embedded tissue sections as described previously.22 We used antibodies against the following: PGP9.5 (1:500; Dako, Glostrup, Denmark), growth associated protein 43 (GAP‐43) (1:1000, Merck, Darmstadt, Germany) and SMAD4 (1:400, Santa Cruz Biotechnology, Dallas, TX, USA). Immunohistochemistry, without the primary antibody, was performed as a negative control.
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