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  • Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries.

Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries.

Nature protocols (2013-10-12)
Sandra G Zimmerman, Nathaniel C Peters, Ariel E Altaras, Celeste A Berg
ABSTRACT

In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)-RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase-conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform.

MATERIALS
Product Number
Brand
Product Description

Roche
T3 RNA Polymerase, from Escherichia coli HB101
Roche
Anti-Digoxigenin-AP, Fab fragments, from sheep
Roche
DIG RNA Labeling Kit (SP6/T7), sufficient for 2 x 10 labeling reactions, kit of 1 (12 components), suitable for hybridization, suitable for Southern blotting
Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Roche
Nylon Membranes, positively charged, roll W × L 0.3 m × 3 m, sheet W × L 10 cm × 15 cm, sheet W × L 20 cm × 30 cm
Sigma-Aldrich
MOPS, ≥99.5% (titration)
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
Sigma-Aldrich
Glycine, BioUltra, for molecular biology, ≥99.0% (NT)
Sigma-Aldrich
Sodium deoxycholate, BioXtra, ≥98.0% (dry matter, NT)
Sigma-Aldrich
Deoxyribonucleic acid sodium salt from salmon testes
Sigma-Aldrich
Sodium phosphate dibasic, BioXtra, ≥99.0%
Sigma-Aldrich
Ethidium bromide, BioReagent, for molecular biology, powder
Sigma-Aldrich
Calcium chloride, anhydrous, BioReagent, suitable for insect cell culture, suitable for plant cell culture, ≥96.0%
Sigma-Aldrich
Sodium phosphate monobasic, BioXtra, ≥99.0%
Roche
NBT/BCIP Stock Solution