Quantification of in situ hybridization signals in rat testes.
We performed basic research into quantifying in situ hybridization (ISH) signals in rat testis, a suitable organ for the quantification because germ cells undergo synchronized development and show stage-specific gene expression. In this model experiment, rRNA was selected as the hybridizable RNA in paraffin sections. Specimens fixed with Bouin's fixative and hybridized with digoxygenin-labeled probes could easily be analyzed quantitatively through "posterization" of the images. The amount of rRNA hybridized with the probe was greatest in early primary spermatocytes, followed by pachytene primary spermatocytes, then diplotene spermatocytes, and finally by secondary spermatocytes and spermatids. The amounts reached low levels in metaphase, anaphase, and telophase of meiotic division and early step 1 spermatids, and then slightly increased during spermiogenesis. ISH rRNA staining was a useful parameter for evaluation of the quantitative analysis of mRNA and the levels of hybridizable RNA in tissue sections.
Kimura
Touji
T
Department of Cytology and Histology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Kosaka
Jun
J
Nomura
Takako
T
Yamada
Teruo
T
Miki
Yukari
Y
Takagi
Koji
K
Kogami
Takashi
T
Sasaki
Junzo
J
D000818:Animals; D017403:In Situ Hybridization; D008297:Male; D012339:RNA, Ribosomal, 28S / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D012671:Seminiferous Tubules / Q000737:chemistry; D013091:Spermatogenesis; D013737:Testis / Q000737:chemistry* / Q000166:cytology
D012339:RNA, Ribosomal, 28S