Five sections of the brain cortex and anterior horns of the spinal cord from each rat were randomly selected and subjected to immunofluorescence staining. The tissue sections were incubated overnight at 4 °C with anti-NF-200 (1:500; Abcam, Cambridge, MA, USA), anti-GAP-43 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), anti-BDNF (1:500; Abcam), anti-NF-κB p65 (1:500; Abcam), Anti-p75NTR (1:500; Abcam), anti-MBP (1:500; Abcam), anti-glial fibrillary acidic protein (GFAP, 1:200, Thermo Fisher Scientific, Waltham, MA, USA), anti-COX-2 (1:1000; BioVision, Milpitas, CA, USA), and anti-CD68 (1:100; Santa Cruz Biotechnology) antibodies, individually. After washing in PBS, the sections were incubated with TRITC/FITC-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. The sections were subsequently mounted on glass slides and coverslipped with Antifade Gel Mount Aqueous Mounting Media (Southern Biotech, Birmingham, AL, USA). Stained sections were viewed under a microscope at 200x magnification, and images were taken of three fields per tissue section. Areas stained positively for GFAP, MBP, and CD68 were analyzed using NIH Image software. The numbers of cells stained positively for GAP43, caspase-3, NF-200, BDNF, NF-kB p65, COX-2, Caspase-3, and p75NTR were counted.
Anti p75ntr
Manufactured by Abcam
Sourced in United States
Anti-p75NTR is a primary antibody that recognizes the p75 neurotrophin receptor (p75NTR). p75NTR is a member of the tumor necrosis factor receptor superfamily and plays a role in neuronal development and apoptosis. This antibody can be used for various applications, including western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Anti nf κb p65, by Abcam (2 mentions)
Anti ngf, by Abcam (2 mentions)
Anti nf200, by Abcam (1 mentions)
Anti gap43, by Santa Cruz Biotechnology (1 mentions)
Anti activated caspase 3, by Cayman Chemical (1 mentions)
Anti glial fibrillary acidic protein, by Thermo Fisher Scientific (1 mentions)
Anti cox 2, by Abcam (1 mentions)
Anti cd68, by Santa Cruz Biotechnology (1 mentions)
Antifade gel mount aqueous mounting media, by Southern Biotech (1 mentions)
Anti bdnf, by Abcam (1 mentions)
Anti mbp, by Abcam (1 mentions)
Anti γ tubulin, by Merck Group (1 mentions)
Anti tap73, by Santa Cruz Biotechnology (1 mentions)
Anti drebrin, by Abcam (1 mentions)
Gapdh, by Beyotime (1 mentions)
Anti psd95, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti p erk, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti p75ntr
1
Immunofluorescence Staining of Rat Brain Tissues
2
Protein Expression Analysis by Western Blot
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Tissue homogenates were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH, 8.0) and subjected to 10% SDS–PAGE. Protein concentrations were determined using the BCA Kit (Thermo). Antibodies used were anti-p75NTR (1:5000; Abcam); anti-TAp73 (1:200, Santa Cruz Biotechnology); anti-PSD95 (1:2000; Abcam); anti-Drebrin (1:1000; Abcam); anti-JNK and anti-phospho-JNK (1:1000; Ruiying Biological); anti-Akt and anti-phospho-Akt (1:1000; Ruiying Biological); anti-γ-tubulin (1:10000; Sigma-Aldrich); GAPDH (1:5000; Beyotime).
3
Quantification of Neuronal Signaling Proteins
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Total proteins prepared from the brain tissue were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h, after which resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was then incubated separately at 4℃ with the following primary antibodies overnight: anti-NGF (Abcam), anti-TrkA (Cell Signaling, Danvers, MA, USA), anti-p-TrkA (Cell Signaling), anti-Akt (Cell Signaling), anti-p-Akt (Cell Signaling), anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-ERK (Santa Cruz Biotechnology), anti-p-75NTR (Abcam), anti-JNK (Cell Signaling), anti-p-JNK (Cell Signaling), anti-Bcl2 (Abcam), anti-Bax (Abcam), and anti-β-actin antibody (Sigma-Aldrich Co.). The membranes were then washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 0.05% Tween 20) and incubated with HRP-conjugated goat anti-rabbit IgG (Invitrogen) and HRP-conjugated goat anti-mouse IgG (Invitrogen) at a 1:1,000 dilution and room temperature for 1 h. Membrane blots were developed using Amersham ECL Select Western Blotting detection reagent (GE Healthcare).
4
Immunofluorescence Analysis of Mouse Brain Sections
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After the mice brain sections (10-μm) cross sections had been blocked (1XPBS, 10% fetal bovine serum, 5% gelatin, and 0.1% Triton X-100) for 1 h, they were incubated with a mixture of two of the following primary antibodies: anti-CD31 (1:100, BD Pharmingen™, San Jose, CA, United States), anti-p75NTR (1:100, abcam, Burlingame, CA, USA ), anti-ZO1 (1:100, abcam, Burlingame, CA, USA), anti-VWF (1:100, abcam, Burlingame, CA, USA ) overnight at 4 °C. For immunochemistry, the brain slides were evaluated by immunochemistry through Dako kits (Dako EnVisionTM+ Dual Link System-HRP, Agilent, CA, USA). The procedure was conducted following the manufacturer’s instruction. The primary antibody was incubated with samples at 4 °C overnight followed by the HRP incubation for 1 h at room temperature. For immunofluorescence staining, the sections after primary antibody incubation were washed with 0.1 M PBS and then incubated with FITC- or Texas Red-conjugated anti-rabbit or anti-mouse IgG (1:400; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature then mounted with antifade mounting medium with DAPI (abcam, Burlingame, CA, USA). The IHC and fluorescence signals were detected and the results recorded using a microscope DMI300B (Leica, Wetzlar, Germany) and captured using Canon EOS550D with EOS Utility.
5
Immunofluorescence Analysis of Atrial Tissue
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At the end of the experiment, the atrial tissue was rapidly excised and fixed in 4% paraformaldehyde at room temperature. Paraffin-embedded tissue was cut into 5 μm sections. Immunofluorescence staining was used to determine the expression and localization of ET-1, NGF, p75NTR, NF-κB p65, and tyrosine hydroxylase (TH) in tissues. Sections were incubated in PBS containing 10% FBS for 60 min and incubated overnight with the primary antibody at 4°C, including anti-ET-1 (ABclonal, China), anti-NGF (Abcam, Cambridge, UK), anti-p75ntr (Abcam, Cambridge, UK), anti-NF-κB p65 (Abcam, Cambridge, UK), and anti-TH (Abcam, Cambridge, UK). Sections were washed with PBS and incubated with secondary antibodies for 1 h at 37°C. After the sections were washed, they were visualized using the DAB reagent. Hematoxylin was counterstained with cores dehydrated with ethanol and sealed with glycerol gelatin. Blinded analysis was performed using Image-Pro Plus 6.0 (Media Cybernetics).
6
Western Blot and Immunofluorescence Assays
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Western blot and immunofluorescence assays were conducted as previously described.34 The antibodies used in Western blot were anti‐GAPDH antibody (1:2000; Immunoway, USA), anti‐Runx2 antibody (1:1000; Abcam, USA), anti‐Collagen1(Col1) antibody (1:2000; Abcam, USA), anti‐β‐catenin antibody(1:1000; CST, USA), anti‐active β‐catenin antibody (1:1000; CST, USA), anti‐phospho‐PI3K antibody (Tyr607, 1:1000; Affinity, USA), anti‐phospho‐Akt antibody (Ser473, 1:1000; Affinity, USA), anti‐PI3K antibody (1:1000; Affinity, USA), anti‐Akt antibody (1:1000; Affinity, USA). The antibodies used in immunofluorescence were anti‐p75NTR (1:200; Abcam, USA) and goat anti‐rabbit IgG‐TRITC secondary antibody (Beyotime, China). The Western blot results were further analysed using BIO‐RAD ChenmiDoc™ XRS + or C‐DiGitTM Blot Scanner from LI‐COR Biosciences. The grayscale analysis was performed with Quantity One software (Bio‐Rad; Hercules, CA, USA).
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