EFFECTS OF HISTOLOGIC AND HISTOCHEMICAL PROCEDURES ON THE INTENSITY OF THE LABEL IN RADIOAUTOGRAPHS OF CELLS LABELED WITH TRITIATED COMPOUNDS
The effects of tissue preparation and extractive procedures on the intensity of radioautographic labeling in terms of mean grain count per cell in cells labeled with tritiated precursors of proteins or nucleic acids were investigated. Ehrlich ascites tumor cells were labeled in vivo with one of three tritiated compounds (thymidine, cytidine, or leucine) and, after 30 min, were examined by radnoautography. Both Carnoy's solution and absolute methanol proved to be reliable fixatives, removing from cells equal amounts of radioactivity. This fixative-soluble radioactivity presumably represents the fraction of precursor that has not been incorporated into nucleic acids or proteins. Treatment with ribonuclease (RNase) or cold perchloric acid (PCA) removed all radioactivity from cells labeled with tritiated cytidine, a precursor of RNA, but not from cells labeled with tritiated thymidine. Extraction in 10% PCA in the cold for 12 to 18 hr gives results comparable to those obtained with RNase, but cells treated with PCA looked better morphologically than cells digested with RNase. Considering that PCA extraction is a much simpler procedure than RNase digestion, PCA extraction as the method of choice for the removal of RNA is proposed. Treatment with deoxyribonuclease removed radioactivity incorporated into DNA, but it also affected to a lesser extent the RNA label. Earlier observation is confirmed that in smears of cells labeled with radioactive leucine the RNA precursors, grains found over the nucleus are not necessarily due to radioactivity incorporated into the nucleus. With leucine, almost half of the grains found over the nuclei of cells in smears are due to radioactivity present in the thin coat of cytoplasm above the nucleus. (BBB)
- Research Organization:
- Northwestern Univ., Chicago
- NSA Number:
- NSA-17-035429
- OSTI ID:
- 4646911
- Journal Information:
- Laboratory Investigation (U.S.), Journal Name: Laboratory Investigation (U.S.) Vol. Vol: 12; ISSN LAINA
- Country of Publication:
- Country unknown/Code not available
- Language:
- English
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