Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization.
The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.
Liscia
D S
DS
Oncogenetics Section, National Cancer Institute, Bethesda, Maryland 20892.
Doherty
P J
PJ
Smith
G H
GH
D000818:Animals; D002364:Caseins / Q000235:genetics*; D004054:Diethylstilbestrol / Q000494:pharmacology; D004853:Epoxy Resins; D005260:Female; D006652:Histological Techniques; D007774:Lactation / Q000378:metabolism; D008297:Male; D008321:Mammary Glands, Animal / Q000032:analysis* / Q000187:drug effects; D008325:Mammary Neoplasms, Experimental / Q000032:analysis; D051379:Mice; D008809:Mice, Inbred C3H; D009693:Nucleic Acid Hybridization; D011247:Pregnancy; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic
D002364:Caseins; D004853:Epoxy Resins; D015347:RNA Probes; D012333:RNA, Messenger; D004054:Diethylstilbestrol