In situ Hybridization for Tyrosinase

Paraformaldehide perfused brains are cryoprotected in 30% sucrose and frozen with cold isopentane.

Frozen brains are cryosectioned at 30 microns in a cryostat

Cryosections are mounted on superfrost slides and dried over-night at room temperature before storing them at -80ºC until processing

Sections are fixed for 10 min in 4% paraformaldehyde

After washing in PBS, sections are permeabilized with Proteinase K for 5 min (1 μg/mL Proteinase K,

50mM Tris·HCl, and 5 mM EDTA)

Refix in 4% paraformaldehyde for 10 min

After rinsing in PBS, proceed with the acetylation step for 10 min with 590

mL of H 2O, 8 mL of triethanolamine, 1 mL of 37% HCl, and 1.5 mL of acetic

anhydride

Incubate for 3 h at room temperature for

prehybridization (50% formamide, 5× SSC, 5× Denhardt’s solution, 250 μg/mL Baker’s yeast RNA, and 500 μg/mL salmon sperm DNA)

Hybridization overnight at 72 °C in

120 μL prehybridization mix

with 2 μL of

digoxigenin-labeled sense (control probe not complementary) and antisense (test

probe complementary) probes for tyrosinase (Riboprobes are generated by PCR and labelled with

the DIG RNA labelling kit from Roche (11175033910))

Wash in 72 °C heated solutions of 5× SSC and 0.2×

SSC, and then equilibrated in B1 buffer [0.1 M Tris·HCl (pH 7.5) and 0.15 M

NaCl]

Preincubation for 1 h in 10% heat-in-activated FCS

in B1 buffer

Incubate overnight with antidigoxigenin-AP Fab

fragments (Roche, diluted 1:5,000 in B1 with 1% heat-inactivated FCS)

Wash in B1 buffer and placed the sections in a

solution of 0.1 M Tris·HCl (pH 9.5), 0.1 M NaCl, and 50 mM MgCl 2 (B2 buffer)

mRNA localization is visualized after overnight incubation with 0.2 mM NBT/BCIP (Roche) and 0.24 μg/mL levamisole in 10% PVA/B2 solution protected from light

The color reaction is terminated by water and mounted in Aquatex (Merck)

Sections are analysed and photographed with a microscope coupled to a camera