Detection of mRNA and hnRNA using a digoxigenin labelled cDNA probe by in situ hybridization on frozen tissue sections.
A full-length cDNA clone encoding the constant region of T cell receptor beta chain was labelled by random priming DNA with digoxigenin-dUTP. The probe was used to estimate the relative amount of the receptor beta chain mRNA by in situ hybridization on frozen sections from human thymus and lymph nodes. The hybridization was visualized in blue using an anti-digoxigenin antibody conjugated with alkaline phosphatase and a subsequent enzyme-catalysed colour reaction. The distributions of the signal in tissue sections were as expected. Moreover, labelled cells showed hybrids both in the cytoplasm and in the nucleus, and strongly and weakly stained cells were clearly distinguishable. The results indicate that this method of in situ hybridization should be useful in the detection of specific mRNA in frozen sections.
Maggiano
N
N
Department of Pathology, Università Cattolica del S. Cuore, Rome, Italy.
Larocca
L M
LM
Piantelli
M
M
Ranelletti
F O
FO
Lauriola
L
L
Ricci
R
R
Capelli
A
A
D002675:Child, Preschool; D015342:DNA Probes; D004076:Digoxigenin; D005629:Frozen Sections; D006801:Humans; D007223:Infant; D008198:Lymph Nodes / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D012332:RNA, Heterogeneous Nuclear / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000737:chemistry; D013950:Thymus Gland / Q000737:chemistry
D015342:DNA Probes; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004076:Digoxigenin