Detection of different mRnas expressed in the thyro-parathyroid complex of the rat by in situ hybridization using digoxigenin-labelled oligonucleotide probes.
The effects have been examined of different methods and regimens for tissue fixation, preservation, permeabilization and immunostaining of different mRNAs detected by in situ hybridization in paraffin-embedded samples. The three main hormone mRNAs expressed in the thyro-parathyroid glands, namely thyroglobulin, calcitonin and parathyroid hormone mRNAs, were chosen as the target nucleic acid sequences to be detected using digoxigenin-labelled probes. Our results suggest that chemical fixation and permeabilization of tissue samples are restrictive steps. Thus, paraformaldehyde fixation provides excellent signal intensities and non-detectable background levels whereas routine formalin and Bouin's solution give unsatisfactory results. A clear linear correlation was also found between signal intensity and proteinase K permeabilization. Moreover, the optimization of immunohistochemical steps, such as anti-digoxigenin antibody concentration and colour development times, enhance the intensity and specificity of hybrid signals. Furthermore, our results show that, in contrast to some data in the literature, paraffin-embedded tissue is suitable for detection of mRNAs by in situ hybridization. It gives equivalent intensities of specific signal and superior histological and cellular resolutions when compared to cryopreserved tissue.
Fernández-Santos
J M
JM
Departamento de Citología e Histología Normal y Patológica, Facultad de Medicina, Universidad de Sevilla, Spain.
Martín-Lacave
I
I
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002116:Calcitonin / Q000235:genetics*; D004076:Digoxigenin / Q000276:immunology; D019286:Endopeptidase K / Q000378:metabolism; D015870:Gene Expression; D017403:In Situ Hybridization / Q000379:methods; D008297:Male; D015345:Oligonucleotide Probes; D010280:Parathyroid Glands / Q000378:metabolism* / Q000473:pathology; D010281:Parathyroid Hormone / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D013950:Thymus Gland / Q000378:metabolism* / Q000473:pathology; D013954:Thyroglobulin / Q000235:genetics*; D016707:Tissue Fixation / Q000379:methods; D014021:Tissue Preservation
D000906:Antibodies; D015345:Oligonucleotide Probes; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D002116:Calcitonin; D013954:Thyroglobulin; D019286:Endopeptidase K; D004076:Digoxigenin