Chapter 12
In Situ Hybridization for RNA: Nonradioactive
Probe: Oligo-DNA Probe: Digoxigenin (1)
YUKAIWASAKIANDIHACHIKATO
Introduction
In this article, protocols are described for preparation of digoxigenin-labeled oligo-DNA probe and their use in in situ hybridization. As has been shown previously (Koji et al. 1988, 1989;
Koji and Brenner 1993; Koji and Nakane 1990, 1996), nonradioactive synthetic oligo-DNA probes can be very useful tools.
The 3' -end of oligo-DNA can be labeled enzymatically with several possible haptens, such as digoxigenin and biotin. Digoxigenin, a plant steroidal aglycone, is especially suitable for immunocytochemical detection because its epitope is not present in animal cells. Also, it has recently been confirmed that under appropriate conditions, there is no substantial difference in the
sensitivity of detection between [ 35 S] cRNA (Denjin et al.
1990) or [ 35 S] cDNA (Unger et al. 1991) probes and nonradioactive probes.
In nonradioactive in situ hybridization, notable improvements have been made in the protocol, including the visualization systems, resulting in greater detection sensitivity. The attempts to quantify colorimetric signals by computer-assisted image analysis are also progressing. (The result shown in this chapter is obtained by the use of an image analyzer.)
Materials
for Prepara- - paraformaldehyde (PF A) (MERCK)
tion of tissues
Correspondence to Yuka Iwasaki, Nagasaki University School of Dentistry,
Department of Periodontology, 1-7-1 Sakamoto, Nagasaki, 852, Japan
(phone +81-95-849-7683; fax +81-95-849-7684)
In Situ Hybridization for RNA: Nonradioactive
Probe: Oligo-DNA Probe: Digoxigenin (1)
YUKAIWASAKIANDIHACHIKATO
Introduction
In this article, protocols are described for preparation of digoxigenin-labeled oligo-DNA probe and their use in in situ hybridization. As has been shown previously (Koji et al. 1988, 1989;
Koji and Brenner 1993; Koji and Nakane 1990, 1996), nonradioactive synthetic oligo-DNA probes can be very useful tools.
The 3' -end of oligo-DNA can be labeled enzymatically with several possible haptens, such as digoxigenin and biotin. Digoxigenin, a plant steroidal aglycone, is especially suitable for immunocytochemical detection because its epitope is not present in animal cells. Also, it has recently been confirmed that under appropriate conditions, there is no substantial difference in the
sensitivity of detection between [ 35 S] cRNA (Denjin et al.
1990) or [ 35 S] cDNA (Unger et al. 1991) probes and nonradioactive probes.
In nonradioactive in situ hybridization, notable improvements have been made in the protocol, including the visualization systems, resulting in greater detection sensitivity. The attempts to quantify colorimetric signals by computer-assisted image analysis are also progressing. (The result shown in this chapter is obtained by the use of an image analyzer.)
Materials
for Prepara- - paraformaldehyde (PF A) (MERCK)
tion of tissues
Correspondence to Yuka Iwasaki, Nagasaki University School of Dentistry,
Department of Periodontology, 1-7-1 Sakamoto, Nagasaki, 852, Japan
(phone +81-95-849-7683; fax +81-95-849-7684)