194
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
of specific mRNA is detected at a cellular level. Our protocol has
proved to be useful for clarifying the expression of mRNA for a
variety of molecules in renal tissues, including, mRNA for complement and its inhibitor (Abe et al. 1996; Sasaki et al. 1997), extracellular matrix and metalloproteinase (Miyazaki et al. 1995).
Our protocol can be modified from a 2-day method to 3-day one.
In that case, sections are incubated with monoclonal anti-DIG
antibody at 4°C overnight (the concentration of the antibody
should be less than that in the 2-day method. On the third
day, immunohistochemistry is performed as described above.
Acknowledgements
The authors thank Drs. A Furusu, K Abe and S Tsukasaki from
the Second Department of Medicine of Nagasaki University and
Drs. R Naka and D Suzuki from Department of Nephrology in
Tokai University for their help. Weare grateful to Professor H
Sakai of Tokai University for his encouragement and support
and Dr. PK Nakane, ex- Professor, Department of Anatomy, Nagasaki University for his advice. Dr. Nikolic-Paterson DJ and
Professor RC Atkins at Monash Medical Center, Melbourne,
Australia for helping us establish the original protocol of in
situ hybridization. We are also grateful to Dr. Takehiko Koji
for his indispensable suggestions.
References
Abe K, Miyazaki M, Koji T, Furusu A, Tsukasaki S, Ozono Y, Harada T,
Nakane PK, Yagame M, Endoh M, Nomoto Y, Sakai H, Kohno S
(1996) Expression of C3 mRNA and DAF mRNA in human diseased kidney. J Am Soc Nephrol 7: 1769.
Furusu A, Miyazaki M, Koji T, Abe K, Ozono Y, Harada T, Nakane PK, Hara
K, Kohno S (1997) Involvement ofinterleukin-4 in human glomerulonephritis. -An in situ hybridization study for IL-4 mRNA and IL-4 receptor
mRNA- J Am Soc Nephrol 8:730-741.
Koji T, Nakane PK (1990) Localization in situ of specific mRNA using thymine-thymine dimerized DNA probes. Sensitive and reliable non-radioactive in situ hybridization. Acta Pathol Jpn 40: 793-807.
Koji T and Nakane PK (1996) Recent advances in molecular histochemical
techniques: In situ hybridization and Southwestern histochemistry.
J Electron Microsc 45:119-127
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
of specific mRNA is detected at a cellular level. Our protocol has
proved to be useful for clarifying the expression of mRNA for a
variety of molecules in renal tissues, including, mRNA for complement and its inhibitor (Abe et al. 1996; Sasaki et al. 1997), extracellular matrix and metalloproteinase (Miyazaki et al. 1995).
Our protocol can be modified from a 2-day method to 3-day one.
In that case, sections are incubated with monoclonal anti-DIG
antibody at 4°C overnight (the concentration of the antibody
should be less than that in the 2-day method. On the third
day, immunohistochemistry is performed as described above.
Acknowledgements
The authors thank Drs. A Furusu, K Abe and S Tsukasaki from
the Second Department of Medicine of Nagasaki University and
Drs. R Naka and D Suzuki from Department of Nephrology in
Tokai University for their help. Weare grateful to Professor H
Sakai of Tokai University for his encouragement and support
and Dr. PK Nakane, ex- Professor, Department of Anatomy, Nagasaki University for his advice. Dr. Nikolic-Paterson DJ and
Professor RC Atkins at Monash Medical Center, Melbourne,
Australia for helping us establish the original protocol of in
situ hybridization. We are also grateful to Dr. Takehiko Koji
for his indispensable suggestions.
References
Abe K, Miyazaki M, Koji T, Furusu A, Tsukasaki S, Ozono Y, Harada T,
Nakane PK, Yagame M, Endoh M, Nomoto Y, Sakai H, Kohno S
(1996) Expression of C3 mRNA and DAF mRNA in human diseased kidney. J Am Soc Nephrol 7: 1769.
Furusu A, Miyazaki M, Koji T, Abe K, Ozono Y, Harada T, Nakane PK, Hara
K, Kohno S (1997) Involvement ofinterleukin-4 in human glomerulonephritis. -An in situ hybridization study for IL-4 mRNA and IL-4 receptor
mRNA- J Am Soc Nephrol 8:730-741.
Koji T, Nakane PK (1990) Localization in situ of specific mRNA using thymine-thymine dimerized DNA probes. Sensitive and reliable non-radioactive in situ hybridization. Acta Pathol Jpn 40: 793-807.
Koji T and Nakane PK (1996) Recent advances in molecular histochemical
techniques: In situ hybridization and Southwestern histochemistry.
J Electron Microsc 45:119-127