RDH5 antibody

Catalog No. ABIN7254061

Product Details anti-RDH5 Antibody

Target: RDH5 (Retinol Dehydrogenase 5 (11-Cis/9-Cis) (RDH5))

Reactivity: Human, Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This RDH5 antibody is un-conjugated

Application: ELISA, Immunohistochemistry (IHC)

Characteristics: Polyclonal Antibody

Purification: Antigen affinity purification

Immunogen: Fusion protein of human RDH5

Isotype: IgG

Application Details

Application Notes: IHC 1:50-1:100, ELISA 1:5000-1:10000

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #104468 (Immunohistochemistry)

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

No.: #104468

Date: 03/23/2023

Antigen: RDH5

Lot Number: SG5433

Method validated: Immunohistochemistry

Positive Control:

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

Negative Control:

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

No primary antibody

Notes:

Passed. Presence of specific signal in the RPE cell layer was considered as indication of specific immunoreactivity using the anti-RDH5 antibody ABIN7254060.

Validation Images

Retinal sections from the wild-type (B6 albino) mice immunostained with anti-RDH5 antibody ABIN7254060. 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.

Full Methods

Primary Antibody: ABIN7254060

Secondary Antibody: 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-RDH5 antibody (antibodies-online, ABIN7254060, lot SG5433) 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.

Experimental 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.

Handling

Format: Liquid

Concentration: 0.84 mg/mL

Buffer: PBS with 0.05 % Sodium azide and 40 % Glycerol, pH 7.4

Preservative: Sodium azide

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

Storage: -20 °C

Storage Comment: Store at -20°C. Avoid freeze / thaw cycles.

References for anti-RDH5 antibody (ABIN7254061)

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).

Target Details for RDH5

Target: RDH5 (Retinol Dehydrogenase 5 (11-Cis/9-Cis) (RDH5))

Alternative Name: RDH5 (RDH5 Products)

Synonyms: 9cRDH antibody, HSD17B9 antibody, RDH1 antibody, SDR9C5 antibody, RDHB antibody, MGC84134 antibody, RDH5 antibody, rdh1 antibody, hsd17b9 antibody, im:6901729 antibody, si:dkey-102c8.5 antibody, 9-cis antibody, AI987873 antibody, RDH4 antibody, cRDH antibody, retinol dehydrogenase 5 antibody, retinol dehydrogenase 1 antibody, retinol dehydrogenase 5 L homeolog antibody, retinol dehydrogenase 5 (11-cis/9-cis) antibody, RDH5 antibody, rdh1 antibody, rdh5.L antibody, rdh5 antibody, Rdh5 antibody

Background: This gene encodes an enzyme belonging to the short-chain dehydrogenases/reductases (SDR) family. This retinol dehydrogenase functions to catalyze the final step in the biosynthesis of 11-cis retinaldehyde, which is the universal chromophore of visual pigments. Mutations in this gene cause autosomal recessive fundus albipunctatus, a rare form of night blindness that is characterized by a delay in the regeneration of cone and rod photopigments. Alternative splicing results in multiple transcript variants. Read-through transcription also exists between this gene and the neighboring upstream BLOC1S1 (biogenesis of lysosomal organelles complex-1, subunit 1) gene.

UniProt: Q92781