RDH11 Antikörper

Produktnummer ABIN966957

Dieses Anti-RDH11-Antikörper ist ein Polyklonal-Antikörper zur Detektion von RDH11 in IHC. Geeignet für Maus. Unabhängig validiert für die Verwendung in Immunohistochemistry.

Kurzübersicht für RDH11 Antikörper (ABIN966957)

Target: RDH11 (Retinol Dehydrogenase 11 (All-Trans/9-Cis/11-Cis) (RDH11))

Reaktivität: Maus

Wirt: Bitte anfragen

Klonalität: Polyklonal

Konjugat: Dieser RDH11 Antikörper ist unkonjugiert

Applikation: Immunohistochemistry (IHC)

Produktdetails anti-RDH11 Antikörper

Anwendungsinformationen

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (IHC)

Validierung #104470 (Immunohistochemistry)

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

No.: #104470

Datum: 23.03.2023

Antigen: RDH11

Chargennummer: 0618

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-RDH11 antibody ABIN966957.

Validierungsbilder

Retinal sections from the wild-type (B6 albino) mice immunostained with anti-RDH11 antibody ABIN966957. 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: ABIN966957   

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-RDH11 antibody (antibodies-online, ABIN966957, lot 0618) 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).
• 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

Lagerung: 4 °C

Details zu RDH11

Target: RDH11 (Retinol Dehydrogenase 11 (All-Trans/9-Cis/11-Cis) (RDH11))

Andere Bezeichnung: RDH11