Aug 05, 2024
In situ Hybridization for Tyrosinase
- Ariadna Laguna1,
- Miquel Vila1
- 1VHIR-CIBERNED-ASAP
- Vilalab Public
- Nuria
External link: https://doi.org/10.1038/s41467-024-53168-7
Protocol Citation: Ariadna Laguna, Miquel Vila 2024. In situ Hybridization for Tyrosinase. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl821w6l2w/v1
Manuscript citation:
Laguna, A., Peñuelas, N., Gonzalez-Sepulveda, M. et al. Modelling human neuronal catecholaminergic pigmentation in rodents recapitulates age-related neurodegenerative deficits. Nat Commun 15, 8819 (2024). https://doi.org/10.1038/s41467-024-53168-7
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 15, 2024
Last Modified: August 05, 2024
Protocol Integer ID: 103428
Keywords: situ hybridization for tyrosinase, situ hybridization, tyrosinase, situ hybridization protocol, labelled riboprobe, situ hybridization protocol with dig
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Abstract
In situ hybridization protocol with DIG-labelled riboprobes
Troubleshooting
Brain sectioning
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
In situ hybridization
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