218
AKIRA MATSUNO ET AL.
Comments
Light microscopic in situ hybridization, which has become a
widely used method for examining the tissue distribution and
expression of mRNA, lacks the ultrastructural spatial relationship between mRNA and the encoded protein. This information,
which can be obtained by electron microscopic in situ hybridization, will be needed to elucidate the intracellular distribution
and role of mRNA for protein synthesis. The major concerns of
electron microscopic in situ hybridization are to maintain tissue
morphology and retain the messages. As Le Guellec et al. stated
(Le Guellec et al. 1992), the ultrastructural preservation in electron microscopic in situ hybridization using ultrathin frozen sections is poor, and specimens embedded in Lowicryl K4M exhibit
poorer ultrastructural preservation than those embedded in
Epon resin. In order to obtain satisfactory morphological preservation, we have routinely utilized 6 ).tm frozen sections fixed
in 4% paraformaldehyde for the preembedding method and
tissues embedded in LR White resin for the postembedding
method.
As described in our previous reports (Matsuno et al. 1994a),
compared with the preembedding method, the postembedding
method presents several difficulties: 1) Difficulty of message preservation during polymerization ofLR White resin at high temperature for an extended period of time, which leads to mRNA
degradation. 2) Relatively high frequency of non-specific signals
because of the non-specific affinity of gold particles. From these
results, it is suggested that the pre embedding method would be
better and easier than the post embedding method because of its
higher sensitivity and better preservation of mRNA.
Our other concern is to visualize mRNA and encoded protein
simultaneously in the same cell. There seem to be two major problems to be resolved in this ultrastructural double staining method. One is to retain the messages and the other is to maintain the
immunoreactivity of the encoded protein in the same cell. As for
mRNA preservation the preembedding electron microscopic in
situ hybridization method using frozen sections fixed in 4% paraformaldehyde has more advantages over the postembedding
electron microscopic in situ hybridization method using tissues
embedded in LR White resin. Frozen sections fixed in 4% paraformaldehyde have better morphological preservation than im-
AKIRA MATSUNO ET AL.
Comments
Light microscopic in situ hybridization, which has become a
widely used method for examining the tissue distribution and
expression of mRNA, lacks the ultrastructural spatial relationship between mRNA and the encoded protein. This information,
which can be obtained by electron microscopic in situ hybridization, will be needed to elucidate the intracellular distribution
and role of mRNA for protein synthesis. The major concerns of
electron microscopic in situ hybridization are to maintain tissue
morphology and retain the messages. As Le Guellec et al. stated
(Le Guellec et al. 1992), the ultrastructural preservation in electron microscopic in situ hybridization using ultrathin frozen sections is poor, and specimens embedded in Lowicryl K4M exhibit
poorer ultrastructural preservation than those embedded in
Epon resin. In order to obtain satisfactory morphological preservation, we have routinely utilized 6 ).tm frozen sections fixed
in 4% paraformaldehyde for the preembedding method and
tissues embedded in LR White resin for the postembedding
method.
As described in our previous reports (Matsuno et al. 1994a),
compared with the preembedding method, the postembedding
method presents several difficulties: 1) Difficulty of message preservation during polymerization ofLR White resin at high temperature for an extended period of time, which leads to mRNA
degradation. 2) Relatively high frequency of non-specific signals
because of the non-specific affinity of gold particles. From these
results, it is suggested that the pre embedding method would be
better and easier than the post embedding method because of its
higher sensitivity and better preservation of mRNA.
Our other concern is to visualize mRNA and encoded protein
simultaneously in the same cell. There seem to be two major problems to be resolved in this ultrastructural double staining method. One is to retain the messages and the other is to maintain the
immunoreactivity of the encoded protein in the same cell. As for
mRNA preservation the preembedding electron microscopic in
situ hybridization method using frozen sections fixed in 4% paraformaldehyde has more advantages over the postembedding
electron microscopic in situ hybridization method using tissues
embedded in LR White resin. Frozen sections fixed in 4% paraformaldehyde have better morphological preservation than im-