RDH10 anticorps

N° du produit ABIN7118460

Détail du produit anti-RDH10 anticorps

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

Reactivité: Humain, Souris, Rat

Hôte: Lapin

Clonalité: Polyclonal

Conjugué: Cet anticorp RDH10 est non-conjugé

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

Purification: Immunogen affinity purified

Pureté: ≥95 % as determined by SDS-PAGE

Immunogène: retinol dehydrogenase 10(all-trans)

Isotype: IgG

Information d'application

Indications d'application: WB: 1:500-1:2000, IP: 1:200-1:1000, IHC: 1:20-1:200

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #104469 (Immunohistochemistry)

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

No.: #104469

Date: 23.03.2023

Antigène: RDH10

Numéro du lot: 20221223E

Application validée: Immunohistochemistry

Contrôle positif:

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

Contrôle négative:

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

No primary antibody

Conclusion:

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

Validation Images

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.

Protocole

Anticorps primaire: ABIN7118460

Anticorps secondaire: 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.

Notes:

• 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.

Stockage

Format: Liquid

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

Agent conservateur: Sodium azide

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

Stock: -20 °C

Stockage commentaire: -20°C for 12 months (Avoid repeated freeze / thaw cycles.)

Date de péremption: 12 months

Références pour anticorps anti-RDH10 (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." dans: Cell reports, Vol. 42, Issue 8, pp. 112982, (2023) (PubMed).

Détails sur RDH10

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

Autre désignation: RDH10 (RDH10 Produits)

Synonymes: anticorps cb804, anticorps rdh10, anticorps SDR16C4, anticorps 3110069K09Rik, anticorps 4921506A21Rik, anticorps AI875664, anticorps AW549993, anticorps D1Ertd762e, anticorps m366Asp, anticorps retinol dehydrogenase 10b, anticorps retinol dehydrogenase 10, anticorps retinol dehydrogenase 10 (all-trans), anticorps rdh10b, anticorps MCYG_00691, anticorps RDH10, anticorps Rdh10

Sujet: 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.

Poids moléculaire: 39 kDa

ID gène: 157506

UniProt: Q8IZV5