13 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
183
signed. The experimenter can also select the sequence, which is
well conserved among different species. Thus, the selected sequence allows the use of one probe on different sections from
different species.
In nonradioactive in situ hybridization, the probe is labeled
with a hapten, such as biotin, digoxigenin (DIG) and thyminethymine (T -T) dimers. The T -T dimers method was originally
described by Drs. Koji and Nakane (1990). The in situ hybridization method using T -T dimerized oligo-probe is described in
-----. Chapter 11. Biotin labeled probe is used in nonradioactive
in situ hybridization, however, endogenous biotin, also called
vitamin H, is present in large quantities in certain organs, including kidney, liver and muscle. Since biotin is inactivated by aldehyde, the presence of biotin in these tissues does not cause a problem when tissues are fixed with aldehyde and embedded in paraffin. While fresh frozen tissues are used for in situ hybridization, blocking of endogenous biotin is an indispensable step
in this method. Endogenous biotin blocking kit is available
from Vector Company. In contrast, since DIG does not exist
in animal and human body, blocking is not necessary, i.e., no
background contamination is present as a result of cross-reaction with endogenous substances.
In this chapter, we describe the protocol of nonradioactive in
situ hybridization using DIG labeled oligonucleotide. The described method is especially suitable for processing renal biopsy
specimens.
Outline
The entire protocol of nonradioactive in situ hybridization with
oligo-DNA probe is as follows:
1. Preparation of the tissue
2. Labeling probe
3. Hybridization with probe
4. Visualization of the bound probe on the section
This outline may be almost similar to other protocols of in situ
hybridization method. The three most important points are as
183
signed. The experimenter can also select the sequence, which is
well conserved among different species. Thus, the selected sequence allows the use of one probe on different sections from
different species.
In nonradioactive in situ hybridization, the probe is labeled
with a hapten, such as biotin, digoxigenin (DIG) and thyminethymine (T -T) dimers. The T -T dimers method was originally
described by Drs. Koji and Nakane (1990). The in situ hybridization method using T -T dimerized oligo-probe is described in
-----. Chapter 11. Biotin labeled probe is used in nonradioactive
in situ hybridization, however, endogenous biotin, also called
vitamin H, is present in large quantities in certain organs, including kidney, liver and muscle. Since biotin is inactivated by aldehyde, the presence of biotin in these tissues does not cause a problem when tissues are fixed with aldehyde and embedded in paraffin. While fresh frozen tissues are used for in situ hybridization, blocking of endogenous biotin is an indispensable step
in this method. Endogenous biotin blocking kit is available
from Vector Company. In contrast, since DIG does not exist
in animal and human body, blocking is not necessary, i.e., no
background contamination is present as a result of cross-reaction with endogenous substances.
In this chapter, we describe the protocol of nonradioactive in
situ hybridization using DIG labeled oligonucleotide. The described method is especially suitable for processing renal biopsy
specimens.
Outline
The entire protocol of nonradioactive in situ hybridization with
oligo-DNA probe is as follows:
1. Preparation of the tissue
2. Labeling probe
3. Hybridization with probe
4. Visualization of the bound probe on the section
This outline may be almost similar to other protocols of in situ
hybridization method. The three most important points are as