Paraffin sections were immersed in 3% hydrogen peroxide for 15 min in order to eliminate the activity of endogenous peroxidase. After nonspecific antigen blocking in 2% bovine serum albumin (BSA), sections were respectively incubated with rabbit anti-CaMK II (1:200, santa cruz), rabbit anti-NMDAR1 (1:400, Epitomics), rabbit anti-GAP-43 (1:300, Cell Signaling Technology) primary antibody at 4°C overnight, then with biotinylated-conjugated anti-rabbit secondary antibody (1:800,Proteintch) for 2 h at 37°C. Followed with avidin-biotin-peroxidase complex (ABC) (1:100, Vector) for 1 h at 37°C. Immunoreactivity was visualized with diaminobenzidine (DAB, Boster Biotech).
Rabbit anti gap 43
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-GAP-43 is a primary antibody that recognizes the growth-associated protein 43 (GAP-43), a protein involved in neuronal growth and development. It is used to detect and quantify the expression of GAP-43 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti camk 2, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti nmdar1, by Abcam (2 mentions)
Mouse anti map2, by Abcam (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Avidin biotin peroxidase complex abc, by Vector Laboratories (1 mentions)
Cryostat microtome, by Leica (1 mentions)
Anti β 3 tubulin, by Cell Signaling Technology (1 mentions)
Fluoro mounting medium, by Merck Group (1 mentions)
Anti mbp, by Cell Signaling Technology (1 mentions)
Alexa fluor 555 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti s100β, by Abcam (1 mentions)
Rabbit anti smad2 3, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho smad2 3, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated anti mouse, by Thermo Fisher Scientific (1 mentions)
Pe conjugated mouse anti human cd90, by BD (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Abcam (1 mentions)
5 protocols using rabbit anti gap 43
1
Immunohistochemical Detection of Neuronal Markers
2
Immunostaining for Neuronal Regeneration
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At 3 or 12 weeks after ONC, tissues were collected after perfusion for immunostaining. Frozen sections (25 μm thickness) were prepared using a cryostat microtome (Leica, Deer Park, IL, USA ) and fixed in 4% PBS-buffered paraformaldhyde solution, and permeabilized using 0.3% Triton-X 100 and 3% goat serum in PBS for immunostaining. Tissue samples were incubated with primary antibodies diluted in PBS that contained 3% goat serum at 4 °C overnight. The primary antibodies used were rabbit anti-GAP43 (1:500, Cell Signaling, Denve, MA, USA), anti-β-III tubulin (1:500, Cell signaling) and anti-MBP (1:500, Cell signaling). After washing 3 times with PBS, the samples were incubated with Alexa Fluor 488 (1:200, green) or Alexa Fluor 555 secondary antibody (1:200, red) (Molecular Probes, Waltham, MA, USA) for 1 h at room temperature. The samples were mounted in fluoro mounting medium (Millipore, Burlington, MA, USA) and images were taken with a Zeiss LSM700 confocal microscope (Kuehnstrasse, Hamburg, Germany). The fluorescence images were analyzed using ImageJ from the National Institute of Health (Bethesda, MD, USA) to analyze the fluorescence intensities of the tissues compared with that of the control after subtracting the background value.
3
Immunocytochemical and Cytometric Analyses
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The following primary antibodies were used for immunocytostaining: rabbit anti-S100β (dilution = 1 : 200) (Abcam, Cambridge, UK), rabbit anti-GAP43 (1 : 200) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-EGR2 (1 : 200) (Abcam), rabbit anti-NCAM (1 : 200) (Sino Biological, Beijing, China), and mouse anti-MAP-2 (1 : 200) (Abcam) antibodies. Alexa Fluor 488-conjugated anti-rabbit (1 : 500) (Life Technologies, Carlsbad, CA, USA) and Alexa Fluor 594-conjugated anti-mouse (1 : 500) (Life Technologies) antibodies were used as secondary antibodies. For western blotting, rabbit anti-Smad2/3 (dilution = 1 : 1000) (Cell Signaling Technology), rabbit anti-phospho Smad2/3 (1 : 5000) (Cell Signaling Technology), and rabbit anti-β-actin (1 : 5000) (Cell Signaling Technology) antibodies were used. For flow cytometric analysis, phycoerythrin- (PE-) conjugated mouse anti-human CD29 (dilution = 1 : 25) (BioLegend, San Diego, CA, USA), Alexa Fluor 488-conjugated rat anti-mouse/human CD44 (1 : 25) (BioLegend), PE-conjugated mouse anti-human CD90 (1 : 5) (BD Biosciences, San Jose, CA, USA), and fluorescein isothiocyanate- (FITC-) conjugated mouse anti-human CD105 (1 : 25) (BioLegend) antibodies were used.
4
Western Blot Analysis of Neuronal Proteins
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Tissues were homogenized in RIPA lysis buffer with 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 1% inhibitor cocktail (Bio Basic Inc). After 30 minutes on ice, the homogenates were centrifuged for 30 min (13,000 g, 4°C). The supernatants were collected for western blot analysis. Total protein concentrations were measuered using the bicinchoninic acid (BCA, Thermo Fisher) method. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently blocked in 5% skim milk for 2 h at room temperature. The membranes were incubated with the following primary antibodies: rabbit anti-CaMK II (1:500, santa cruz), rabbit anti-NMDAR1 (1:1000,Epitomics), rabbit anti-GAP-43 (1:1000, Cell Signaling Technology), mouse anti-β-actin (1:4000; aBCAm) for overnight, then with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:4000, Proteintch) and anti-mouse secondary antibody (1:4000,Proteintch) for 2 h at room temperature.The immunopositive bands were detected by using an enhanced chemiluminescent substrate (Thermo Fisher) and a Bio-Rad ChemiDoc XRS digital documentation system (Bio-Rad). The band density was quantified using Image J software. The amount of protein expression is presented relative to the levels of β-actin.
5
Immunolabeling of Neural Markers
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The following antibodies were used as primary antibodies for immunocytostaining and for immunohistochemistry: rabbit anti‐s100b (dilution = 1:1,000) (Dako, Ely, UK), rabbit anti‐p75 (1:500) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti‐GAP43 (1:200) (Cell Signaling Technology), rabbit anti‐glial fibrillary acidic protein (GFAP) (1:200) (Abcam, Cambridge, UK), rabbit anti‐NG2 (1:1,000) (Millipore, Cambridge, UK), mouse anti‐myelin basic protein (MBP) (1:100) (Chemicon, Chandlers Ford, UK), goat anti‐protein zero (P0) (1:500) (Abcam), rabbit anti‐Tuj‐1 (1:100) (Chemicon), mouse anti‐neurofilament (NF) (1:1,000) (Sigma), rabbit anti‐PGP9.5 (1:500) (Abcam), mouse anti‐fibronectin (Millipore), mouse anti‐Nanog (1:200) (ReproCELL, Yokohama, Japan), mouse anti‐MAP‐2 (1:200) (Abcam), and rabbit anti‐NeuN (1:500) (Abcam) antibodies. As secondary antibodies, Alexa Fluor 546‐conjugated anti‐rabbit and anti‐mouse antibodies and Alexa Fluor 670‐conjugated anti‐rabbit and anti‐mouse antibodies (1:1,000) (Life technologies, Carlsbad, CA, USA) were used.
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