Fig. 4. Immunolocalization of 3 and 7 nAChRs in chemotaxing KCs. Colonies of second-passage human foreskin KCs grown on coverslips inserted underneath the agar gel of the chemotaxis AGKOS plates (Fig. 1B) were fixed to avoid cell-membrane permeabilization. (A) Dual immunolabeling with antibody to nAChR subunit and ß1 integrin analysed by deconvolution microscopy. The images show cell-membrane colocalization (yellow) of 3 or 7 nAChR subunit (green) with the integrin ß1 (red). The images of nAChR subunits and ß1 integrin were acquired in a single plane. Bar, 30 µm. (B) Immunostaining with the 3- or 7-specific rabbit antibodies before and 45 minutes and 90 minutes after addition of 1 mM choline to the chemoattractant well. The vertical arrow indicates the position of the chemoattractant well. Notice that the haphazard pattern of receptor distribution on the cell membrane of intact KC changes after exposure to a chemoattractant. The 7 receptor accumulated at the leading edge (lamellipodium), decorating the filopodia (anterior cytoplasmic spikes). The 3 receptor immunoreactivity was abundant at the frontal cell area behind the leading edge. Specific staining was eliminated when the primary rabbit anti-receptor antibody was omitted or when the rabbit antiserum was preincubated with the peptide used for immunization. No immunostaining was observed when the KCs were treated with pre-immune sera obtained from the same rabbits (data not shown). Bar, 25 µm.