Adapted from Seydoux, G. and Fire, A. (1995). Whole-mount
in situ hybridization for the detection of RNA in
C. elegans embryos. In
C. elegans: Modern Biological Analysis of an Organism. Methods in Cell Biology (ed. H. Epstein and D. Shakes) Academic Press, San Diego.
I. Materials
A. Reagents
- Anti-digoxigenin Fab fragment, rhodamine labeled (Boehringer Mannheim Cat# 1207 750)-
Anti-digoxigenin Fab fragments, alkaline-phosphatase labeled. (Boehringer Mannheim Cat# 1093 274)-
BSA (Fraction V, Sigma Cat# A-7906)-
Commercial Bleach (5.25% Sodium hypochlorite solution, Clorox)-
DAPI (Sigma Cat# D-1388)-
DNA from salmon testes (Sigma, Cat# D-1626) -
Formaldehyde (37%, Fisher, Cat# F79-500)-
Formamide (Boehringer Mannheim Cat# 100 731)-
Glycerol (Boehringer Mannheim Cat#100 649)-
Glycine (Sigma Cat# G-4392)-
Glycogen (Boeringher Mannheim Cat# 901-393)-
Heparin (Sigma, Cat# H-3393)-
Hepes (Boehringer Mannheim Cat# 242 608)-
Levamisole (Sigma, Cat# L-9756) -
NaN3 (Sigma Cat# S-2002)-
NBT: 4-Nitro blue tetrazolium chloride. (Boehringer Mannheim Cat# 1383 213)-
p-Phenylenediamine (Sigma Cat# P-6001)-
0.1% Poly-L-lysine solution (Sigma Cat# P-8920). -
Polyoxyethylene-Sorbitan Monolaurate (Tween 20; Sigma Cat# P-1379)-
Proteinase K (Boehringer Mannheim, Cat# 161 519)-
Taq DNA polymerase (Promega Cat# M1861)-
Tris (Boehringer Mannheim Cat# 604 205, 812 854)-
Triton X-100 (Sigma Cat# X-100) -
X-phosphate: 5-Bromo-4-chloro-3-indolyl-phosphate. (Boehringer Mannheim Cat# 1383 221)
B. Stock solutions
DEPC treatment of solutions is not needed. Solutions are stored at -20oC unless otherwise indicated.-
10x dNTP mix: Digoxigenin-11-dUTP pre-mixed with other nucleotides (1 mmol/l dATP, 1 mmol/l dCTP, 1 mmol/l dGTP, 0.65 mmol/l dTTP, 0.35 mmol/l DIG-dUTP). (Boehringer Cat# 1277 065.) -
10x PBS: 80g NaCl, 2g KCl, 6.1g anhydrous Na2HPO4, 2g KH2PO4, H2O to 1 liter. Autoclave and store at room temperature.-
10x Taq Buffer: 500 mM KCl, 100 mM Tris-HCl (pH 9.0 at 25oC), 1% Triton X-100. -
20x SSC: 3 M NaCl, 0.3 M Na3Citrate-2H20. Store at room temperature.-
Mounting media: 70% glycerol, 1mg/ml p-phenylenediamine (pH 9).
C. Working solutions
These solutions are prepared on the day of use.-
Formaldehyde fixative solution: 1X PBS, 0.08 M Hepes (pH 6.9), 1.6 mM MgSO4, 0.8 mM EGTA, 3.7% formaldehyde.-
Hybridization buffer for cDNA-derived probes: 100 µg/ml autoclaved salmon sperm DNA, 50 µg/ml heparin, 0.1% Tween 20, 50% formamide, 5 X SSC.
[ If you are using Wheaton jars that hold up to 20 slides in 150 ml, you will need a total of 1 liter of HYB buffer for the pre-HYB and post HYB washes:
Prepare 1 liter of HYB buffer omitting the DNA.
Aliquot HYB into 4 bottles as follows:
- 400ml in one bottle labeled HYB w/DNA. Add 4ml of 10mg/ml ssDNA to that aliquot.
- 360ml in one bottle labeled HYB wash
- 180ml in one bottle labeled 3:2. Add 120ml of PTw to that aliquot.
- 60ml in one bottle labeled 1:4. Add 240ml of Ptw to that aliquot.]-
Hybridization buffer for oligonucleotide probes: 100 µg/ml autoclaved salmon sperm DNA, 50 µg/ml heparin, 0.1% Tween 20, and appropriate concentrations of formamide and SSC as described in section III D.-
Hypochlorite solution: 1N NaOH, 1:10 dilution of commercial bleach. -
PBT: 1 x PBS, 0.1% BSA, 0.1% Triton X-100-
PTw: 1 x PBS, 0.1% Tween 20-
Staining solution: 100 mM NaCl, 5 mM MgCl2, 100 mM Tris, pH 9.5; 0.1% Tween 20; 1mM Levamisole. Levamisole is a potential inhibitor of endogenous phosphatases.-
TTBS: 150mM NaCl, 50mM Tris-HCl pH 7.8, 0.1% BSA, 0.1% Tween-20
E. Slides and other materials
- Carter's rubber cement (Dennison Stationary Products).-
Coverslips for freeze-cracking (No. 11/2; 24 x 50mm; Thomas Scientific Cat# 6663K94)-
Incubation dishes. Unless otherwise noted, all washes and incubations are done in Wheaton staining dishes (Thomas Cat# 8541-H15), which can hold 20 slides in 150 ml.-
Parafilm squares cut to 20 x 20 mm. -
Slides (75 x25mm) [Cel-Line Associates Inc. (tel: 1-800-662-0973), Cat# 10-2066, brown autoclavable coating]. These slides have 3 square wells (14x14mm) surrounded by a thin hydrophobic coating similar in thickness to a C. elegans embryo. This coating supports the coverslip during freeze-cracking and facilitates incubation with small volumes of staining solutions. Only the two outside wells are used. Wells are subbed with poly-lysine on the day of use: 50 µl of poly-lysine solution is allowed to settle on slides for 10-20 min; excess solution is wiped off and slides are baked at 60oC for 10 min.
II. PROCEDURE
A. Overview.
A mixed population of C. elegans embryos is attached to microscope slides, permeabilized by freezing and fixed with methanol and formaldehyde. Embryos are then incubated overnight with a digoxigenin-labelled single-stranded DNA probe, followed by extensive washes to remove excess probe. Fluorescent or enzyme-linked anti-digoxigenin antibodies are used to visualize the hybridized probe. The entire procedure requires approximately 1.5 days from harvest of embryos to probe visualization.
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