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. 2021 Sep 17;9:730550. doi: 10.3389/fcell.2021.730550

FIGURE 3.

FIGURE 3

Detection of specific HAPLN–lectican molecular interactions by an in situ proximity ligation assay (PLA). In situ PLA to visualize in situ endogenous HAPLN–lectican interactions (green) in the medial nucleus of the trapezoid body of 5-month-old adult WT (A–D) and KO (E–G) mice using VGLUT1 as a marker of the calyx of Held (magenta). A pair of primary antibodies of HAPLN4 and brevican shows dot signals at the edge or perisynaptic space of the calyx of Held terminals (C, arrowheads). Notably, in the absence of HAPLN4, brevican shows an ectopic shift of localization to the surrounding neuropil, where HAPLN1 might potentially stabilize brevican in the aberrant condition (F, arrowheads). The specificity of the PLA reaction is validated with two different negative controls: one is a reaction that omits the anti-HAPLN4 antibody on wild-type tissue (D); the other is the PLA reaction of HAPLN4 and brevican using Hapln4-KO tissue (G). The non-specific reactions in the negative controls are indicated by asterisks (D,G). Acan, aggrecan; Bcan, brevican. The scale bar represents 10 μm.