Instructions For Use
Standard bone marrow or peripheral blood smears may be used for immunofluorescence staining. Cytocentrifuge preparations of cell suspensions made up in RPMI-1640 tissue culture medium containing 10% foetal calf serum are also suitable. Frozen sections should be cut as thin as possible. Store unfixed slide preparations in a dessicator at room temperature. 1.
Circle two areas of interest (1 cm diameter) with diamond pencil on the slide to be stained. One will be used as a control area and the other is for the test. Fix smears for 30 min. in absolute methanol at 4o
C immediately before staining. Air dry.2.
Block by adding a drop of normal goat serum to both circled areas. Let stand for 5 min. Rinse off goat serum with PBS. 3.
Apply 25 ul rabbit anti human TdT antibody mixture to the test area and 25 ul of negative control to control area and allow to react for 30 min in a humid chamber. 4.
Rinse avoiding cross-contamination of reagents. Now wash for 10 min. in a Coplin jar with several changes of PBS. Agitate occasionally for thorough washing.5.
Wipe the slide, avoiding cells, and shake excess wash solution off circled area. Spread 25 ul of FITC goat anti rabbit IgG F(ab')2
and incubate in humid chamber for 30 min in the dark. 6.
Rinse off and wash for 10 min. in a Coplin jar with several changes of PBS.7.
Apply small drops of glycerol/PBS (mounting medium) to two glass cover slips and invert circled areas on slide over dropped cover slips. 8.
Examine by epifluorescence and phase microscopy. Evaluate the percentage of nucleated cells exhibiting nuclear fluorescence.