RDH10 Antikörper

Produktnummer ABIN7118460

Produktdetails anti-RDH10 Antikörper

Target: RDH10 (Retinol Dehydrogenase 10 (All-Trans) (RDH10))

Reaktivität: Human, Maus, Ratte

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser RDH10 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunohistochemistry (IHC), ELISA, Immunoprecipitation (IP)

Aufreinigung: Immunogen affinity purified

Reinheit: ≥95 % as determined by SDS-PAGE

Immunogen: retinol dehydrogenase 10(all-trans)

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise: WB: 1:500-1:2000, IP: 1:200-1:1000, IHC: 1:20-1:200

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (IHC)

Validierung #104469 (Immunohistochemistry)

by: Palczewski Lab, Center For Translational Vision Research, UC Irvine

No.: #104469

Datum: 23.03.2023

Antigen: RDH10

Chargennummer: 20221223E

Validierte Anwendung: Immunohistochemistry

Positivkontrolle:

Retina cryosection from B6 Albino (B6(Cg)-Tyrc-2J/J) animal

Negativkontrolle:

Retina cryosection from B6 Albino (B6(Cg)-Tyrc-2J/J) animal

No primary antibody

Bewertung:

Passed. Presence of specific signal in the RPE cell layer was considered as indication of specific immunoreactivity using the anti-RDH10 antibody ABIN7118460.

Validierungsbilder

Retinal sections from the wild-type (B6 albino) mice immunostained with anti-Rdh10 antibody ABIN7118460. DAPI staining shows localization of the inner (INL) and outer (ONL) nuclear layer of the mouse retina. Cralbp (Rlbp1) co-staining was used to visualize RPE and Müller glia cells in the retina. Presence of specific signal in the RPE cell layer confirms specific immunoreactivity.

Protokoll

Primärantikörper: ABIN7118460

Sekundärantikörper: donkey anti-rabbit AF647-conjugated antibody (Abcam, 150075)

Full Protocol:

• Collect eyes from mice and fix with paraformaldehyde 4% (Electron Microscopy Sciences, 15710) in 1x PBS for 30 min at RT.
• Cryoprotection with sucrose series:
• • Wash in 10% sucrose in 1x PBS.
  • Immerse in 10% sucrose in 1x PBS for 30 min at RT.
  • Wash in 20% sucrose in 1x PBS.
  • Immerse in 20% sucrose in 1x PBS for 30 min RT.
  • Wash in 30% sucrose in 1x PBS.
  • 30% sucrose ON at 4°C.
• Embed eyes in OCT compound (Tissue-Tek O.C.T. Compound, 4583).
• Cut retinal sections at a thickness of 12 μm on a cryostat.
• Air dry sections for 15 min at RT, store at -80°C until use.
• Bring sections to RT and rehydrate in 1x PBS for 1 h.
• Incubate sections in blocking buffer (1x PBS, 3% BSA (Sigma-Aldrich, A7030), 3% Donkey serum (Sigma-Aldrich, S30-100ML), 0.1% Triton X-100 (Sigma-Aldrich, X100-500ML)) for 1 h at RT.
• Incubate sections with primary rabbit anti-RDH10 antibody (antibodies-online, ABIN7118460, lot 20221223E) diluted 1:50 in blocking buffer ON at RT. Include a no primary antibody negative controls. Additionally, counterstaing with primary mouse anti-CRALBP antibody (Thermo Fisher Scientific, MA1-813).
• Rinse sections 3 times with 1x PBS, 0.1% Triton X100. Keep negative controls in a separate container.
• Incubate sections with secondary AF647-conjugated donkey anti-rabbit antibody (Abcam, Ab150075) or AF488-conjugated donkey anti-mouse antibody (Thermo Fisher Scientific, A32766) diluted 1:500 in blocking buffer for 1 h at RT.
• Rinse sections once with 1x PBS, 0.1% Triton X-100 for 5 min at RT.
• Incubate sections in 1x DAPI (Thermo Fisher Scientific, 62248) in 1x PBS, 0.1% Triton X-100 for 15 min at RT.
• Rinse sections 3x with 1x PBS, 0.1% Triton X-100 for 5 min at RT.
• Mount sections in VECTASHIELD® HardSet™ Antifade Mounting Medium (Vector Laboratories, H-1400) mounting medium.
• Acquire images with a fluorescence microscope and appropriate filter settings.For the validation purposes Keyence BZ-X800E fluorescence microscope was used with following filters: BZ-X DAPI for DAPI, BZ-X GFP for AF488, BZ-X Cy5 for AF647. Images were taken at 10x and 40x magnification.

Anmerkungen:

• Experiment involved validation of the specificity of 4 antibodies against different Rdh proteins: Rdh5 (ABIN7254060), Rdh10 (ABIN7118460), Rdh11 (ABIN966957), and Rdh12 (ABIN7167836). All 4 proteins are important for eye function and highly expressed in neural retina and/or RPE. Validation is based on comparison of each staining with known pattern of expression in the mouse retina. For review highlighting each Rdh localization see PMID20801113.

• To aid orientation in the cell layers anti-Cralbp counterstain was included in the staining (Thermo MA1-813). Cralbp (Rlbp1) is highly expressed in RPE and Müller glia cells in mouse retina.

Handhabung

Format: Liquid

Buffer: PBS with 0.02 % sodium azide and 50 % glycerol pH 7.3,

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Lagerung: -20 °C

Informationen zur Lagerung: -20°C for 12 months (Avoid repeated freeze / thaw cycles.)

Haltbarkeit: 12 months

Referenzen für anti-RDH10 Antikörper (ABIN7118460)

Tworak, Kolesnikov, Hong, Choi, Luu, Palczewska, Dong, Lewandowski, Brooks, Campello, Swaroop, Kiser, Kefalov, Palczewski: "Rapid RGR-dependent visual pigment recycling is mediated by the RPE and specialized Müller glia." in: Cell reports, Vol. 42, Issue 8, pp. 112982, (2023) (PubMed).

Details zu RDH10

Target: RDH10 (Retinol Dehydrogenase 10 (All-Trans) (RDH10))

Andere Bezeichnung: RDH10 (RDH10 Produkte)

Hintergrund: Synonyms:SDR16C4 Background:Retinol dehydrogenase with a clear preference for NADP. Converts all-trans-retinol to all-trans-retinal. Has no detectable activity towards 11-cis-retinol, 9-cis-retinol and 13-cis-retinol.

Molekulargewicht: 39 kDa

Gen-ID: 157506

UniProt: Q8IZV5