Immunostaining with anti-Lnα4 was not detected in the extrasynaptic BM of muscle fibers from ALS EOMs (
Fig. 1B2–B3). In contrast, strong labeling with anti-Lnα4 was found in the vast majority of NMJs in all ALS EOMs (
Fig. 2B,
2F), with the exception of donor 2/RL, where weakly labeled or unlabeled NMJs accounted for more than half of the NMJs examined (
Fig. 2B3–B4;
Table 4). Strong staining with anti-Lnα4 was found frequently in NMJs where immunostaining with anti-Lnα2 was not observed, in donor 3/RS+RL and donor 4/RL (
Table 4;
Fig. 2E). The localization of Lnα4 labeling varied in different NMJs and between donors, in contrast to the constant staining pattern seen with anti-Lnα5 antibody at NMJs (see below). Thus, we examined five additional EOM specimens with 213 new NMJs, in addition to the 350 NMJs presented in
Table 3, to fully understand the distribution of laminin α4 chain at NMJs in ALS. Three distinct staining patterns with anti-Lnα4 were identified after double labeling with α-bungarotoxin in cross sections. Lnα4 was present (1) at both pre- and postsynaptic sites in the majority of NMJs (
Fig. 2F1), as also typically seen in controls, (2) only at presynaptic sites (
Fig. 2F2), and the least common (3) only at postsynaptic sites (
Fig. 2F3). It was often seen that the BMs of terminal Schwann cells and axons in the presynaptic region were strongly stained with anti-Lnα4 in the first case (
Fig. 2F4). Staining with anti-Lnα4 was strong in both peri- and endoneurium of intramuscular nerve bundles (
Fig. 1B2). However, less stained peri- or endoneurium plus disrupted BM of peri- and endoneurium were occasionally encountered (
Fig. 1B3). The blood vessel staining was weak or moderately, whereas the capillaries were, in general, moderately stained by anti-Lnα4 (
Fig. 1B2).