Subcellular localization of sTNFR1-tm and sTNFR1-tm-Y by immunofluorescence microscopy. Indirect immunolocalization of the TNF-R and a marker for secretory lysosomes, lgp120. The cells used were RBL cells transfected with sTNFR1-tm-Y (A-D), sTNFR1-tm (E-H), and wild-type RBL cells (I-L). The cells were fixed, permeabilized, and stained, as described in “Materials and methods,” before being attached to poly-lysine–coated coverslips and the images recorded. Briefly, the cells were incubated with a rabbit anti–lgp120 antibody and a mouse monoclonal antibody against the sTNFR1. Thereafter, the cells were stained with Alexa Fluor–labeled anti–rabbit and anti–mouse secondary antibodies. The localization of TNF-R and lgp120 is shown in red and green, respectively, whereas the rightmost images show an overlay of the red and green staining patterns. The corresponding Nomarski images also are shown. Results are representative of 4 separate experiments. Bar = 10 μM.