Biotin dextran

Here, we generated different multivalent formulations of a given pMHC specificity to enable comparative analysis. In order to ascribe the observed differences to the multimerization scaffold, all the multivalent pMHC formulations (tetramer, dextramer and spheromer) were made using the same stock of purified MHC molecules. The pMHC-tetramers were generated as described previously(63 (link)). Briefly, fluorophore-conjugated streptavidin (Invitrogen) was added to each pMHC monomer incrementally to achieve a 4:1 (pMHC:SAv) molar ratio. Next, streptavidin agarose was added to each tetramer for quenching any unbound, biotinylated pMHC. After filtration, biotinylated agarose beads were added to remove any unsaturated streptavidin molecules. The protein was filtered and stored at 4°C until further use. We also used a previously described protocol for generating the pMHC-dextramers(19 (link)). The biotinylated pMHC molecules were incubated with fluorophore-conjugated streptavidin (Invitrogen) at a molar ratio of ~3.5:1 (pMHC:SAv) for 30 mins at room temperature. To this mixture, biotin-Dextran (MW = 70kDa, Thermo Fisher Scientific) was added at a molar ratio of ~30:1 (pMHC:Dextran) and incubated further for another 30 mins at room temperature. The spheromer assembly has already been described above.

Motor neurons supplying individual muscles were retrogradely labeled using 0.1% CTB-555, 10% 3000 MW Tetramethylrhodamine-dextran or 10% 3000 MW Dextran-biotin (Molecular probes) in vivo as described (Demireva et al., 2011 (link); Sürmeli et al., 2011 (link)). Biotin was detected using Streptavidin-488 (Molecular probes).
P0–p4 mice were anaesthetized on ice and a small incision was performed on the skin to expose the muscle of interest. Dye was loaded into pulled glass microelectrodes by suction and injected into the muscle through application of positive pressure. Dextran injections were performed at multiple sites, whereas CTB injections were performed at a single site. For motor neuron visualization, animals were sacrificed 1–2 days following dye injection.
E14.5 embryos were eviscerated and dissected in ice-cold aCSF (127 mM NaCl, 3 mM KC1, 1.25 mM NaH₂P0₄, 26 mM NaHC0₃, 10 mM D-glucose, 1 mM MgCl₂ and 2 mM CaCl₂). The embryos were pinned and the dorsal epidermis was removed. A pulled glass capillary was used to inject 10% Tetramethylrhodamine Dextran or Dextran Biotin into individual muscles. After injection, embryos were incubated in continuously oxygenated circulating aCSF (95/5% CO₂/O₂) for 4–5 hours at 27°.