We previously described the intracellular localization of murine mammary
tumor virus (MuMTV) p28 protein in thin sections (1). In that study, MuMTV
containing cells fixed in 3% paraformaldehyde plus 0.05% glutaraldehyde were
labelled after thin sectioning using ferritin-antiferritin in an unlabelled
antibody technique. We now describe the labelling of murine leukemia virus
(MuLV) particles using the unlabelled antibody technique coupled to
ferritin-Fab antiferritin.
Cultures of R-MuLV in NIH/3T3 cells were grown to 90% confluence (2),
fixed with 2% paraformaldehyde plus 0.5% glutaraldehyde in 0.1 M cacodylate
at pH 7.2, postfixed with buffered 17 OsO4, dehydrated
with a series of etha-nols, and embedded in Epon. Thin sections were
collected on nickel grids, incubated in 107
H2O2, rinsed in HEPES buffered
saline, and subjected to the immunoferritin labelling procedure. The
procedure included preincubation in 27 egg albumin, a four hour incubation
in goat antisera against purified gp69/71 of MuLV (3) (primary antibody),
incubation in F(ab’)2 fragments of rabbit antisera to
goat IgG (secondary antibody), incubation in apoferritin, incubation in
ferritin-Fab ferritin, and a brief fixation with 2% glutaraldehyde. The
sections were stained with uranyl acetate and examined in a Siemens IA
electron microscope at an accelerating voltage of 60 KV.
