One well from each hemisphere of all birds was double-labeled for parvalbumin (PV) and chondroitin sulfate, one of the main components of the perineuronal nets, following a previously described protocol [22 (link), 37 (link), 41 (link)]. Briefly, sections were blocked in 5% Normal Goat Serum (NGS) diluted in Tris-buffered Saline (TBS) with 0.1% Triton-X-100 (TBST) for 30 minutes. They were incubated overnight at 4°C in a mixture of 2 primary antibodies diluted in TBST: a mouse monoclonal anti-chondroitin sulfate antibody (clone CS-56, 1:500 for Experiment 1 and 3 or 1:1000 for Experiment 2, C8035, Sigma Aldrich) specific for the glycosaminoglycan portion of the chondroitin sulfate proteoglycans that are the main components of the PNN and a polyclonal rabbit anti-parvalbumin antibody (1:1000; ab11427, Abcam). Sections were then incubated at room temperature in a mixture of secondary antibodies diluted in TBST. A goat anti-mouse IgG coupled with Alexa 488 (green, 1:100, Invitrogen) was used to visualize PNN staining and a goat anti-rabbit IgG coupled with Alexa 546 (red, 1:200, Invitrogen) was used to visualize PV cells. Finally, sections were mounted on slides using TBS with gelatin and coverslipped with Vectashield containing DAPI (H-1500, Vector laboratories) to confirm that PNN that were not surrounding PV-positive cells were localized around a cell nucleus.
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