FIXATION CONDITIONS FOR DNA AND RNA IN-SITU HYBRIDIZATION - A REASSESSMENT OF MOLECULAR MORPHOLOGY DOGMA

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choic e for in situ hybridization (ISH), and specific anecdotal cautions int erdicting the use of precipitating fixatives, which otherwise may offe r certain advantages such as superior nuclear detail, are common, Few systematic studies addressing ISH fixation conditions have been publis hed, We reasoned that heavy metals present in some precipitating fixat ives may compromise duplex formation during ISH, Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) an d two negative cell lines (K562 and MOLT 4) were expanded to > 10(10) and pellets feued in NBF, zinc formalin, B5, and Bouin's and Hollande' s solutions, and subjected to DNA ISH using biotinylated genomic probe s, Ten tissue biopsies fixed in. both Hollande's and NBF solutions wer e also evaluated for human papilloma virus content using DNA ISH. Addi tionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with flu orescein isothiocyanate-labeled oligonucleotides, Catalyzed reporter d eposition combined with Streptavidin-Nanogold staining and silver acet ate autometallography (Catalyzed reporter deposition-Ng-autometallogra phy ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals en trapped in fixed tissues were removed by two exposures to Lugol's iodi ne, Results for both DNA and RNA ISH comparing B5 and NBF fixatives we re virtually identical. Hollande's, Bouin's, B5, and zinc formalin fix ed tissue showed results indistinguishable from NBF fixed tissue in DN A ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.