Sc 74516

Manufactured by Santa Cruz Biotechnology

Sourced in Germany

Sc-74516 is a laboratory product manufactured by Santa Cruz Biotechnology. It is a research-grade tool designed for use in scientific applications. The core function of Sc-74516 is to facilitate specific laboratory procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Lsm 880 confocal microscope, by Zeiss (3 mentions) Ab150108, by Abcam (1 mentions) Evostm xl core imaging system, by Thermo Fisher Scientific (1 mentions) Lsm 880, by Zeiss (1 mentions) Alexa fluor 555 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti mouse igg2a, by Thermo Fisher Scientific (1 mentions) Alexa fluor 647 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions) Dapi fluoromount g mounting medium, by Southern Biotech (1 mentions) Mab397, by Merck Group (1 mentions) S3023, by Agilent Technologies (1 mentions) Ab52818, by Abcam (1 mentions) Phalloidin ifluor 647 reagent, by Abcam (1 mentions) Ep1553y, by Abcam (1 mentions) Af938, by R&D Systems (1 mentions)

4 protocols using sc 74516

Cochlear Hair Cell Morphology Analysis

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Cochlear whole mounts (two rats per group) were prepared to examine the morphology of the outer hair cells of the cochlea [27 (link),28 (link)]. The cochleae were dissected, and the otic capsule bone was decalcified. Free-floating, dissected cochlear outer hair cells were subjected to immunofluorescent staining. The primary antibodies 1:1000 anti-myosin 7A (Sc74516; Santa Cruz) were incubated overnight at 4 °C. The secondary antibodies 1:2000 Alexa 594 anti-mouse IgG (ab150108; Abcam) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were incubated for 2–3 h. The cochlear tissues were mounted on slides and imaged using a confocal microscope (Zeiss LSM 880; Zeiss, Land Baden-Württemberg, Germany).
H&E staining (n = 2 rats per group) was performed to examine the organ of Corti and spiral ganglion cells [29 (link),30 (link)]. Dissected cochleae were embedded in paraffin blocks, and the cochlear blocks were sectioned at a thickness of 10 µm. The slides were stained with H&E solutions (hematoxylin for 5 min and eosin for 45 s). The stained slides were examined using the EVOS^TM XL Core Imaging System (#AMEX1000; Invitrogen, Carlsbad, CA, USA).

Lee C.H., Jeon J., Lee S.M, & Kim S.Y. (2022). Pravastatin Administration Alleviates Kanamycin-Induced Cochlear Injury and Hearing Loss. International Journal of Molecular Sciences, 23(9), 4524.

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Immunohistochemistry of Cochlear Hair Cells

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Animals were transcardially perfused with 4% paraformaldehyde in 0.1 M PBS following flushing with prewarmed PBS. The cochleae of the guinea pigs were removed and immersed in 4% paraformaldehyde for 1 h at RT. The organ of Corti was carefully dissected and permeabilized with 5% horse serum in PBS supplemented with 0.3% Triton X-100 for 1 h. The samples were then incubated with anti-CtBP2 IgG1 (1:100; 612044, BD Biosciences, San Jose, CA, USA), anti-GluA2 IgG (1:1000; MAB397, Merck KGa, Darmstadt, Germany) and anti-myosin 7a (1:100; sc-74516, Santa Cruz Biotechnology Inc., Dallas, TX, USA) polyclonal antibodies with 3% horse serum in PBS supplemented with 0.3% Triton X-100 overnight at 37 °C. After three washes with PBS, the tissues were incubated with secondary antibodies against CtBP2 (Alexa Fluor™ 555 goat anti-mouse IgG1, 1:500, A21127, Thermo Fisher Scientific, Waltham, MA, USA), GluA2 (Alexa Fluor™ 488 goat anti-mouse IgG2a, 1:1000, A21131, Thermo Fisher Scientific, Waltham, MA, USA), and myosin 7a (Alexa Fluor™ 647 donkey anti-rabbit IgG (H+L), 1:500, A31573, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37 °C. The samples were mounted with DAPI Fluoromount-G^® mounting medium (SouthernBiotech, Birmingham, AL, USA) and covered with a coverslip for analysis. Images were obtained using an LSM 880 Zeiss confocal microscope.

Lin Y.C., Shih C.P., Lin Y.Y., Lin H.C., Kuo C.Y., Chen H.K., Chen H.C, & Wang C.H. (2024). C-Phycocyanin Attenuates Noise-Induced Cochlear Synaptopathy via the Inhibition of Oxidative Stress and Intercellular Adhesion Molecule-1 in the Cochlea. International Journal of Molecular Sciences, 25(10), 5154.

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Immunofluorescent Staining of Cochlear Hair Cells

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After washing twice with PBS and the cochlear explants fixed with 4% paraformaldehyde for 20 minutes. The explants were permeabilized in 0.5% Triton X-100, blocked in 5% normal goat serum (NGS), and incubated overnight with the primary antibody against myosin 7a (1:100, sc-74516, Santacruz) at 4°C. Explants were then washed three times with NGS and incubated for 1.5 hours with Alexa Fluor 564 goat anti-mouse secondary antibody (1:200, A11005, Invitrogen). After washing with NGS and distilled water, explants were counterstained with DAPI for 5 minutes and briefly washed with distilled water. Finally, explants were dehydrated in the air and mounted with fluorescence mounting medium (S3023, Agilent Technologies, CA, USA). Images of the stained middle-turn cochlear explants were observed with a Zeiss LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany).
For Annexin V staining, permeabilized explants were washed with PBS and binding buffer and were incubated for 10 minutes with buffer containing Annexin V-FITC in the dark. The procedure of myosin 7a staining and the observation of fluorescent images were performed as described above. The numbers of myosin 7a and annexin V positive inner hair cells (IHCs) and outer hair cells (OHCs) were counted over a distance of 160 um in the middle turn of each cochlear explant.

Kang B.C., Yi J., Kim S.H., Pak J.H, & Chung J.W. (2023). Dexamethasone treatment of murine auditory hair cells and cochlear explants attenuates tumor necrosis factor-α-initiated apoptotic damage. PLOS ONE, 18(9), e0291780.

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Cochlear Immunohistochemistry in Mice

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Cochleae were dissected from newborn (postnatal day 0, P0), P6, P12, P25 and P90 mice. After rapid decapitation, inner ears were removed and fixed for 2h in 4% formaldehyde. If necessary, tissues were decalcified prior to cryosectioning. Sections were cut parallel to the modiolus (mid-modiolar cut). Tissue sections of the cochlea were incubated overnight at 4 °C with primary antibodies directed against LDLR (rabbit monoclonal antibody [EP1553Y]; 1:50; Abcam; ab52818), myosin VIIa (mouse monoclonal antibody; 1:50; Santa Cruz Biotechnology; sc-74516), Kir4.1 (mouse monoclonal antibody; 1:50; Santa Cruz Biotechnology; sc-293252) and VE-cadherin (goat polyclonal antibody; 1:50; R&D Systems; AF938). The anti-LDLR antibody used in this study was found to give a positive signal in western-blot performed with liver extracts of wildtype mice, but not with liver extracts of Ldlr -/ -mice ( Li et al., 2021 ) (link). Tissues were then incubated for 1h with Rhodamine Red X-, Cy5-and/or FITC-conjugated anti-mouse, anti-goat and antirabbit IgGs secondary antibodies (Jackson Immunoresearch Laboratories). F-actin staining was obtained using Phalloidin-iFluor 647 reagent (1:50; Abcam; ab176759). Antibody specificity was tested by omitting each of the primary antibodies. Pictures were representative of three age-matched cochleae, from three different animals.

Saume A., Thiry M, & Defourny J. (2021). LDLR expression in the cochlea suggests a role in endolymph homeostasis and cochlear amplification. Hearing research, 409.

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