126
HIDEFUMI YOSHIOKA AND SUMIHARE NOTI
Results
While using this protocol, dense accumulation of signal grains
indicating expression of the epidermal growth factor (EGF) gene
was observed in the granular convoluted tubules of a mouse submandibular gland, as shown in Fig. 2 (Noji 1990).
Troubleshooting
• Problem 1. Sections peel offfrom a slide glass.
Remedy: Check surface treatment of slide glasses.
• Problem 2. Emulsion layer peels off from a slide glass.
Remedy: Clean the surface of the slide glasses with xylene before coating it with emulsion.
• Problem 3. RNA probe is not synthesized.
Remedy: Check type of RNA polymerase or linearlized sites.
Check purity of template DNA.
• Problem 4. High background.
Remedy: Check probe size. Check emulsion and developing
chemicals (Use fresh). Check safelights.
• Problem 5. Positive control is negative.
Remedy: Check fixation processes. Check probes.
References
Noji S., Takahashi N., Nohno T., Koyama E., Yamaai T., Muramatsu M., and
Taniguchi S (1990) Expression pattern of the homeobox gene Hox-3.5
during mouse development, as revealed by a simplified in situ hybridization method. Acta Histochem. Cytochem. 23:353-366.
Noji S. (1997) A practical use of a kit for in situ hybridization with tissue
section. In: Immunostaining and In situ hybridization (Y odosha The
Protocol Series). Yodosha, Tokyo, pp58-79
Angerer LM. and Angerer RC (1992) In situ hybridization to cellular RNA
with radiolabelled RNA probes. In: Wilkinson DG (ed) In situ hybridization, IRL press, Oxford New York Tokyo, pp15-32
HIDEFUMI YOSHIOKA AND SUMIHARE NOTI
Results
While using this protocol, dense accumulation of signal grains
indicating expression of the epidermal growth factor (EGF) gene
was observed in the granular convoluted tubules of a mouse submandibular gland, as shown in Fig. 2 (Noji 1990).
Troubleshooting
• Problem 1. Sections peel offfrom a slide glass.
Remedy: Check surface treatment of slide glasses.
• Problem 2. Emulsion layer peels off from a slide glass.
Remedy: Clean the surface of the slide glasses with xylene before coating it with emulsion.
• Problem 3. RNA probe is not synthesized.
Remedy: Check type of RNA polymerase or linearlized sites.
Check purity of template DNA.
• Problem 4. High background.
Remedy: Check probe size. Check emulsion and developing
chemicals (Use fresh). Check safelights.
• Problem 5. Positive control is negative.
Remedy: Check fixation processes. Check probes.
References
Noji S., Takahashi N., Nohno T., Koyama E., Yamaai T., Muramatsu M., and
Taniguchi S (1990) Expression pattern of the homeobox gene Hox-3.5
during mouse development, as revealed by a simplified in situ hybridization method. Acta Histochem. Cytochem. 23:353-366.
Noji S. (1997) A practical use of a kit for in situ hybridization with tissue
section. In: Immunostaining and In situ hybridization (Y odosha The
Protocol Series). Yodosha, Tokyo, pp58-79
Angerer LM. and Angerer RC (1992) In situ hybridization to cellular RNA
with radiolabelled RNA probes. In: Wilkinson DG (ed) In situ hybridization, IRL press, Oxford New York Tokyo, pp15-32