At days 0, 1, 3 and 7, animals were euthanized with a urethane intraperitoneal injection (1 g/kg), and both temporal bones were immediately removed. Two cochleae were homogenized in a tube containing ice-cold lysis buffer (100 mM Tris, pH 7.4, 200 mM NaCl, 1% NP-40, 10 mM MgCl2) with protease inhibitors. Western blotting was performed, as described previously.17 (link) In brief, lysates were centrifuged for 20 min at 13 000 r.p.m. at 4 °C; in addition, proteins in the supernatant were separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were incubated overnight with the primary antibodies against 4-HNE (1 : 1000 dilution), BDNF (ab6201, 1 : 1000 dilution; Abcam) and NT-3 (ab65804, 1 : 1000 dilution; Abcam). After rinsing in TBST, the membranes were incubated with the HRP-conjugated secondary antibody (81–1620, A16104, G21040; 1 : 5000; Invitrogen) for 1 h. Bands were detected by chemiluminescence (LF-QC0103, Ab Frontier, Seoul, Korea). Further, blots were stripped and reprobed with actin (SC-1616, Santa Cruz Biotechnology), thereby serving as a protein loading control. Band intensities were quantified using the ImageJ software (NIH).
Ab6201
Manufactured by Abcam
Sourced in United States
Ab6201 is a primary antibody that recognizes the human CD3 epsilon protein. It is intended for use in immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab65804, by Abcam (1 mentions)
G 21040, by Thermo Fisher Scientific (1 mentions)
Lf qc0103, by AbFrontier (1 mentions)
Sc 1616, by Santa Cruz Biotechnology (1 mentions)
Ab3523, by Abcam (1 mentions)
Application suite, by Leica (1 mentions)
Dfc290, by Leica (1 mentions)
Ab16053, by Abcam (1 mentions)
Ab177487, by Abcam (1 mentions)
Ab10062, by Abcam (1 mentions)
D9542, by Merck Group (1 mentions)
Sc 547, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Sod 101, by Stressgen (1 mentions)
Sc 32886, by Santa Cruz Biotechnology (1 mentions)
Sc 30147, by Santa Cruz Biotechnology (1 mentions)
Ecl film, by Cytiva (1 mentions)
Sod 110, by Stressgen (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
4 protocols using ab6201
1
Temporal Bone Protein Analysis
2
Quantifying BDNF and iNOS in Rat Brain Post-Lesion
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On Day 14, rats were anesthetized with isoflurane and perfused transcardially with phosphate-buffered solution. Brains were removed, fixed in 10% formalin for one week, dehydrated in a graded ethanol series, embedded in paraffin, and processed for a paraffin block which was cut in 6µm thick sections. For immunostaining, the sections were de-paraffinized in xylene, rehydrated in a graded series of ethanol, washed with normal saline and microwaved in citrate buffer for 10 min. Hydrogen peroxide was used to block endogenous peroxidase activity which enhanced nonspecific immunoreactivity. Two antibodies, brain-derived neurotrophic factor (BDNF, ab6201, 1/50, Abcam, Cambridge, MA, USA) and inducible nitric oxide synthase (iNOS, ab3523, 1/20, Abcam, Cambridge, MA, USA) were used to evaluate the upregulation of neural plasticity and antioxidant effects, respectively.21 (link),22 (link) Immunostained sections were digitized using a 40 × objective (Leica DFC 290, Leica, Heerbrugg, Germany) in conjunction with the Leica Application suite (version 3.3.0, Leica). Five views from each area of the peri-lesional site were assessed by a pathologist blinded to the experimental conditions (S.W.C.) and stained cells were counted and averaged.
3
Immunohistochemistry Analysis of Neuronal Markers
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Every one-tenth of the continuous sections of each sample was used to perform immunohistochemistry. Briefly, after being washed with PBS, the sections were penetrated by 1% Triton X-100 for 1 h and incubated with blocking buffer at room temperature (RT) for 1 h. Subsequently, the tissues were incubated with primary antibodies overnight at 4°C, fluorescent 488- or 568-conjugated secondary antibodies (1 : 400) for 2 h at RT, and 4′,6-diamidino-2-phenylindole (DAPI, 1 : 2000, Sigma, D9542) for 2 min. Finally, the sections were coverslipped with Vectashield (Vector). The following primary antibodies were used: mouse anti-glial fibrillary acidic protein (GFAP, 1 : 500, Abcam, ab10062), rabbit anti-neuron-specific nuclear protein (NeuN, 1 : 400, Abcam, ab177487), rabbit anti-brain-derived neurotrophic factor (BDNF, 1 : 400, Abcam, ab6201), rabbit anti-nerve growth factor (NGF, 1 : 200, Millipore, 04-1119) or rabbit anti-neurotrophin-3 (NT-3, 1 : 200, Santa Cruz, SC-547) for spinal cord sections, and rabbit anti-growth-associated protein 43 (GAP-43, 1 : 500, Abcam, ab16053) for sciatic nerve sections.
4
Hippocampal Protein Quantification
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The hippocampus was homogenized in 0.05 M sodium phosphate buffer (pH 6.65). Subsequently, the protein concentration was determined using BCA method (Thermo Scientific Pierce, USA), described by Stich [30 (link)]. CuZn SOD (SOD1), Mn SOD (SOD2), CAT, GPx, GR, BDNF, TH, DAT, and COMT proteins were assayed by Western blot analysis as described previously by Gavrilović et al. [27 (link)]. Antibodies used for the quantification of specific proteins were as follows: SOD1 (SOD-101, Stressgen, USA), SOD2 (SOD-110, Stressgen, USA), CAT (Calbiochem, Germany), GPx (sc-30147 Santa Cruz Biotechnology, USA), GR (sc-32886, Santa Cruz Biotechnology, USA), BDNF (ab6201, Abcam, USA), TH (ab51191, Abcam, USA), DAT (ab18548, Abcam, USA), and β-actin (ab8227, Abcam, USA). After washing, the membranes were incubated in the secondary anti-rabbit (dilution 1 : 5000, Amersham ECL™ Western Blotting Analysis System, UK) antibodies conjugated to horseradish peroxidase. A secondary antibody was then visualized by the Western blotting-enhanced chemiluminescent detection system (ECL, Amersham Biosciences, UK). The membranes were exposed to ECL film (Amersham Biosciences, UK). The result was expressed in arbitrary units normalized in relation to β-actin, which is in accordance with our previous protocol [27 (link)].
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