Demonstration of terminal deoxynucleotidyl transferase in single cells by indirect immunofluorescence--a methodological reappraisal.
The optimal conditions for assaying terminal transferase by indirect immunofluorescence with commercially available reagents have been explored. Target tissues examined included fresh, stored and cultured human acute lymphoblastic leukemia cells; normal human peripheral blood and bone marrow; and calf thymus. Brief, cold fixation in glutaraldehyde/ethanol or omission of fixation proved to be satisfactory. The most suitable primary antibody was an unfractionated rabbit serum with specificity for anti-calf thymus TdT which cross-reacted with human material and was neutralized in high dilution by purified enzyme. Inferior reagents and techniques gave false negative results. Identification of TdT appears to be feasible even in tissue samples stored for several months and enumeration of cells is facilitated by ethidium bromide counterstaining.
D000818:Animals; D000906:Antibodies; D001853:Bone Marrow / Q000201:enzymology; D002478:Cells, Cultured; D003429:Cross Reactions; D004253:DNA Nucleotidylexotransferase / Q000032:analysis* / Q000276:immunology; D004254:DNA Nucleotidyltransferases / Q000032:analysis*; D004996:Ethidium; D005069:Evaluation Studies as Topic; D005188:False Negative Reactions; D005455:Fluorescent Antibody Technique; D006801:Humans; D007945:Leukemia, Lymphoid / Q000201:enzymology; D051381:Rats; D013194:Staining and Labeling; D013950:Thymus Gland / Q000201:enzymology
D000906:Antibodies; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase; D004996:Ethidium