Materials 21 General

1. Diethylpyrocarbonate- (DEPC) treated water. DEPC (Sigma, Poole, UK) is added to double-distilled water to a concentration of 0.1% (v/v), the solutions are shaken, allowed to stand for 1 h, and then autoclaved.

2. The following equipment is required: a dissection microscope offering up to at least 65x magnification (e.g., Nikon, Kingston upon Thames, UK or Zeiss,

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

Fig. 1. (A) In situ hybridization of Fgf-8 to a stage 20 chicken embryo using a mouse cDNA sequence as probe. (B) The same probe as in (A) hybridized to a 9.5-d mouse embryo. (C) In situ hybridization of a chicken Fgf-3 probe to a stage 11 chicken embryo. (D) Vibroslice (thick, 50 |im) section of a chicken embryo hybridized with a ret probe showing transcripts in the lateral regions of the otic vesicles. (E) Thin (10 |im) section of the specimen in (C), showing Fgf-3 hybridization to the neural tube and branchial pouch endoderm. (F) "Two-color" in situ hybridization of Krox-20 (red) and Hox-B1 (blue) showing expression in adjacent segments (rhombomeres) of the embryonic zebrafish hindbrain. (G) A typical gel showing DIG-labeled riboprobes before (lane 1) and after (lanes 2 and 3) DNase treatment. Note that the intensities of the probe (lower) bands are much greater than that of the DNA template (upper) bands. Lane M is a DNA mol-wt standard ladder. Note that the RNA in lanes 1-3 appears as a "doublet" even though the enzymes used to linearize the template do not have 3'- overhangs. Whatever the source of the "doublet," we do not find that it affects the use or sensitivity of such probes. (See color plate 12 appearing after p. 368.)

Fig. 1. (A) In situ hybridization of Fgf-8 to a stage 20 chicken embryo using a mouse cDNA sequence as probe. (B) The same probe as in (A) hybridized to a 9.5-d mouse embryo. (C) In situ hybridization of a chicken Fgf-3 probe to a stage 11 chicken embryo. (D) Vibroslice (thick, 50 |im) section of a chicken embryo hybridized with a ret probe showing transcripts in the lateral regions of the otic vesicles. (E) Thin (10 |im) section of the specimen in (C), showing Fgf-3 hybridization to the neural tube and branchial pouch endoderm. (F) "Two-color" in situ hybridization of Krox-20 (red) and Hox-B1 (blue) showing expression in adjacent segments (rhombomeres) of the embryonic zebrafish hindbrain. (G) A typical gel showing DIG-labeled riboprobes before (lane 1) and after (lanes 2 and 3) DNase treatment. Note that the intensities of the probe (lower) bands are much greater than that of the DNA template (upper) bands. Lane M is a DNA mol-wt standard ladder. Note that the RNA in lanes 1-3 appears as a "doublet" even though the enzymes used to linearize the template do not have 3'- overhangs. Whatever the source of the "doublet," we do not find that it affects the use or sensitivity of such probes. (See color plate 12 appearing after p. 368.)

Welwyn, Garden City, UK); transmitted (Nikon, Zeiss) and fiber optic (Schott, Zeiss, Leica, Milton Keynes, UK) light sources; heated water bath and microfuge (e.g., Eppendorf, Heraeus) for enzyme digests and riboprobe synthesis (e.g., Grant, Merck, Poole, UK); horizontal gel electrophoresis tank and power supply

(e.g., Life Technologies, Paisley, Scotland); oven (e.g., Heraeus, Merck), water bath or Spin 'n' Stack incubator (Hybaid, Teddington, UK) for hybridizations and washes; rotating wheel or shaker at 4°C and at room temperature for incubations with antibodies and subsequent washes; suitable photomicroscopes and equipment for histological analysis. For sectioning a vibrotome and/or microtome are required.

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