ATS

Anti-Melanopsin Protocol

as seen in Targeting Trends Newsletter, Jul-Aug-Sep 2009 and Oct-Nov-Dec 2009

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We’re interested in trying out your melanopsin antibody (Cat. #AB-N38) using immunohistochemistry in mouse retina. Do you have a recommended protocol?

Yes. The following protocol has been utilized successfully with anti-melanopsin.

Panda S. et al. 2002. Melanopsin (Opn4) requirement for normal light- induced circadian phase shifting. Science 298(5601):2213-2216.


Immunostaining Protocol (AB-N38)
* corrected August 19, 2009 *

Remove the corneas, and postfix eyes at 4°C for 24 hours in 4% paraformaldehyde in phosphate-buffered saline (PBS). Remove lenses.

Cryoprotect eyecups for sectioning at 4°C for 24 hours in 30% sucrose in PBS; embed the eyecups in OCT medium (Sakura Finetek, Torrance, CA), freeze, section (16-20 µm), and thaw-mount onto gelatin-coated slides.

Dissect retinas destined for flat-mounting from eyecups immediately after postfixation, stretch onto filter paper, and process in 1.5 ml microfuge tubes.

Wash tissue (slides and flat-mounts) 3 times (10 min, 4°C) in Tris- buffered saline (TBS, Quality Biological, Gaithersburg, MD) and block for 30 min at 4°C in 1.5% normal goat serum in TBS.

Incubate tissue for 24 hr at 4°C in a 1:2,500 dilution of antiserum UF006 in a TBS-incubating buffer containing 1% bovine serum albumin, 0.25% carrageenan lambda and 0.003% Triton X-100.

Wash slides and flat mounts three times in TBS (10 min, 4°C) and incubate for 1 hour at 22°C in Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:500 in TBS incubating buffer.

Wash 3 final times in TBS (10 min, 22°C).

Remove flat-mounts from the filter paper and transfer onto glass microscope slides. Mount Flat-mounts and sections in DAPI-containing Vectashield (Vector Laboratories, Burlingame, CA), coverslip, and seal with clear fingernail polish.