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Aldh1a3

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ALDH1A3 is an enzyme that catalyzes the oxidation of aldehydes to carboxylic acids. It is involved in the metabolism of retinol and other aldehydes.

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2 protocols using aldh1a3

1

Quantification of ALDH Isoforms

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Cells were washed with PBS and solubilized in PRO-PREPTM protein extraction solution (iNtRON biotechnology, Korea) for 15 min at 4°C. Cell debris was eliminated by centrifugation at 14,000 rpm for 10 min at 4°C. The supernatant was analyzed for protein content by the Bradford method, and 20 μg of protein was separated on a 10% polyacrylamide gel. The protein was transferred to a hydrobond ECL nitrocellulose membrane (Amersham Biosciences; Piscataway, NJ) and incubated overnight at 4°C with polyclonal antibody against ALDH1A1 (Abcam; Cambridge, MA; 1:1000 dilution), ALDH1A2 (Abcam; Cambridge, MA; 1:1000 dilution), ALDH1A3 (Genetex; Cambridge, MA; 1:1000 dilution), or ALDH3A1 (Cusabio; CA; 1:1000 dilution) in Tris-buffered saline (50 mM Tris, pH 7.5, 150 mM NaCl) with 0.05% polysorbate-20 containing 5% skim milk. After washing three times for 10 min with Tris-buffered saline with Tween 20, the membrane was incubated with secondary antibodies for 1 h at room temperature. Immune reactive protein was detected with an enhanced chemiluminescence kit and quantified using Quantity One software (Bio-Rad Laboratories, CA).
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2

Immunohistochemical Analysis of LSCD

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Paraffin-embedded tissue sections (9 months after the LSCD was induced) were used to conduct immunohistochemical analysis. Tissue sections were stained using ALDH1A1 (R&D Systems, Inc. Minneapolis, MN, USA), ALDH1A3 (GeneTex, Inc. Irvine, CA, USA) and Pax-6 (Millipore Sigma—Life Science, St Louis, MI, USA) antibodies. Alexa Fluor® 488 donkey anti-mouse fluorophore conjugated secondary antibodies was used. Cell nuclei staining was performed using propidium iodide (Invitrogen, Eugene, OR, USA). Slides were analyzed using a Nikon 400 fluorescent microscope. Picture processing and analysis was performed using Adobe Photoshop CS5 (Adobe, New York, NY, USA).
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