Cochlear samples were prepared using a procedure similar to that used for the histological hair cell analysis. The decalcified samples of cochlea were rinsed in 10 mM PBS containing 10% and 30% sucrose, embedded in Tissue-Tek O.T.C. Compound (Sakura Finetechnical), and frozen in liquid nitrogen. Thin sections (8 μm) were obtained with a cryostat (CM3000, Leica Instruments). Immunohistochemistry was performed according to a previously described procedure with modification42 (link). Briefly, tissue sections were rinsed in 0.1% Triton X-100/Tris buffered saline (TBS), blocked with 3% bovine serum albumin/0.3% Triton X-100/TBS (TBST) for 30 min at room temperature, and incubated with an unconjugated AffiniPure Fab fragment anti-mouse IgG (1:10, Jackson ImmunoResearch) for 2 hr at room temperature. The sections were incubated overnight with primary antibodies (anti-4HNE; 1:400, JaICA. anti-Myosin 7a; 1:500, Abcam) diluted in 0.3% TBST at 4 °C. All of the sections were washed three times in 0.1% TBST and incubated with secondary antibodies (Cy3 anti-mouse; 1:500, Jackson ImmunoResearch, Alexa Fluor 488 anti-rabbit; 1:500, Jackson ImmunoResearch) for 1 hr at room temperature under light-protected conditions. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:2,000).
Anti myosin7a
Manufactured by Abcam
Sourced in United Kingdom
Anti-myosin7a is a primary antibody that specifically recognizes the myosin7a protein. Myosin7a is a member of the myosin superfamily and is involved in the process of intracellular transport and organelle movement.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Alexa fluor 488 anti rabbit, by Jackson ImmunoResearch (1 mentions)
Anti mouse cy3, by Jackson ImmunoResearch (1 mentions)
Cm 3000, by Leica (1 mentions)
Polyvinylidene fluoride transfer membrane, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Lysis buffer, by iNtRON Biotechnology (1 mentions)
Alexa 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti myosin7a
1
Immunohistochemical Analysis of Cochlear Samples
2
Myosin 7a Protein Expression in Mouse Cochlea
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Two cochlea tissues were dissected from right and left temporal bone of P5 mice and homogenized in lysis buffer (iNtRON Biotechnology, Seongnam, Gyeonggi, Korea). The samples were centrifuged at 20,000 g for 30 min after sonication. Then 30 mg protein was boiled with 1x sample buffer for 5 min and loaded on 6–10% SDS polyacrylamide gel. After running at 100 V for 3 hr, the proteins were transferred to polyvinylidene fluoride transfer membrane (Millipore, Darmstadt, Hesse, Germany) at 250 mA for 2 hr. The membrane was blocked with 10% skim milk at room temperature for 1 hr and incubated at 4℃ overnight with primary antibody: anti-myosin7a (1:100, Abcam, Cambridge, Cambridgeshire, UK) or anti-Beta actin (1:10,000, Sigma, Saintlouis, Missouri, USA). Next it was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, anti-rabbit (1:2,500, Cell signaling technology, Danvers, Massachusetts, USA), and anti-mouse (1:50,000, Novus biologicals, Littleton, Colorado, USA) at room temperature for 1 hr and then proteins were detected by ECL (GE healthcare, Waukesha, Wisconsin, USA).
3
Whole Mount Immunofluorescence of Cochlea
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For whole mount immunofluorescence of cochlea, temporal bones of littermate were obtained at 5 days after birth and fixed in 4% paraformaldehyde at 4℃ overnight. The organ of Corti were dissected from temporal bones. Samples were incubated in 5% Triton-X for 30 min and blocked with 1X PBS containing 5% horse serum and 3% BSA for 1 hr. Tissues were stained at 4℃ overnight by anti-myosin7a (1:250, Abcam, Cambridge, Cambridgeshire, UK). Next, the samples were washed in 1X PBS at 10 times for 15 min and incubated overnight at 4℃ with goat-anti-IgG Alexa 488 secondary antibody (1:300, Abcam, Cambridge, Cambridgeshire, UK) and Alexa 594 Phalloidin (1:250, Life Technologies, Carlsbad, California, USA). Finally, the tissues were washed with 1X PBS at 10 times for 15 min and mounted with antifade on glass slides. Pictures of outer hair cells (OHCs) and inner hair cells (IHCs) were taken from parts of the apex, middle, and base of the cochlea duct using confocal microscopy (LSM 710; Carl Zeiss, Oberkochen, Baden-Württemberg, Germany). Then the intensity of primary antibody was measured using a ZEN program.
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