In order to study the incidence of Estrogen Receptors (ER) in breast carcinoma, lung carcinoma and melanoma, an in situ hybridisation technique for ER mRNA (ER mRNA-ISH) was developed. Various technical aspects of the procedure including tissue fixation, hybridisation conditions, and demonstration technique were investigated in order to obtain an optimum technique for routine use. ISH results were compared with ER immunohistochemistry using the monoclonal antibodies ER1D5 and D5. Commercially available biotin labelled antisense oligonucleotides to ER, Poly A (total mRNA), and sense chromogranin (negative control) were applied to frozen and formalin-fixed paraffin sections of breast carcinomas. For frozen sections, various fixatives including formalin, alcohol, Schoobridge, Zamboni's and acetic- alcohol were compared. A direct streptavidin- eroxidase and an indirect demonstration method using anti-biotin were also compared. The effect of differing formamide concentrations and post hybridisation stringency washings were analysed. An optimised ISH technique was then applied to frozen sections of 21 cases of breast carcinoma and 11 cases of lung carcinoma. Results were compared to H222 staining on adjacent sections. The ISH technique was also optimised for use on formalin-fixed, paraffin-embedded sections of 28 breast carcinomas and 17 melanomas. The results were compared with ER1D5 and D5 immunohistochemistry done on adjacent sections. The occurrence of endogenous biotin was also studied on a range of normal tissues. Consistent ISH results were obtained when formamide was omitted from the hybridisation cocktail, high stringency post hybridisation washes were discarded, room temperature hybridisations and an indirect demonstration method were used. Fixation of frozen sections in acetic/ethanol gave more consistent results with good morphology and resulted in positive nucleolar staining in 90% of breast and 45% of lung carcinomas. Positive nucleolar staining was also present in frozen sections of one metastatic melanoma. In formalin fixed paraffin sections, acid hydrolysis and pronase treatment were required prior to ISH. Cytoplasmic and/or nucleolar ER mRNA-ISH staining was seen in 87% of breast carcinoma and 97% of melanoma studied. ER1D5 was present in 54% of breast carcinomas but was absent in all melanomas. D5, on the other hand, was found in 88% of the melanomas. In conclusion, ER mRNA-ISH can be successfully done on acetic/alcohol fixed frozen sections and formalin fixed paraffin sections. Formamide, high stringency washes and elevated hybridisation temperatures are detrimental to a successful ISH reaction and an indirect demonstration method (using anti-biotin) is preferred. Unfortunately, endogenous biotin can cause false positive ISH reactions and needs to be considered during interpretation. Results show that the localisation of ER mRNA in the nucleolus is specific. Both ER mRNA-ISH and ER immunohistochemistry indicate that melanomas and some lung carcinomas contain a receptor possibly similar to that in breast carcinomas. / Thesis (M.Sc.)--Department of Anatomical Sciences, 2004.
| Identifer | oai:union.ndltd.org:ADTP/280140 |
| Date | January 2004 |
| Creators | Henwood, Anthony F |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_US |
| Detected Language | English |
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