Fig. 8.

Co-localisation of alpha1, alpha2 and beta3 subunits in individual synapses on CA1 pyramidal cell somata. Sequential applications of two antibodies with different size immunoparticles for the two subunits in each replica. The antibody labelled with 5-nm particles was applied first. Extrasynaptic subunit labelling is marked by vertical arrows for the 5-nm particles and horizontal arrows for the 10-nm particles. (A) Two synapses (1, 2) are labelled for both alpha1 (5 nm, e.g. arrowheads) and beta3 (10 nm, large vertical arrows) subunits. (B) Two synapses (1, 2) are labelled for both the alpha2 (5 nm, e.g. arrowheads) and beta3 (10 nm, large vertical arrows) subunits. (C) Two synapses (1, 2) are labelled for both the alpha1 (5 nm, e.g. arrowheads) and alpha2 (10 nm, large vertical arrows) subunits. The ratio of labelling for the two subunits is different in the two synapses (synapse 1, alpha1/alpha2, 2/12 particles; synapse 2, alpha1/alpha2, 14/16 particles). Note that labelling intensity differences between antibodies do not represent differences in subunit abundance, because the degree of labelling is not comparable between antibodies due to the individual properties of each antiserum. The application of a primary antibody applied second in the sequence and the use of larger particles both reduce labelling efficacy. Scale bar: 0.2 μm (A–C).