Immunohistochemical GABA Staining

Sections were first deparaffinized using a sequence of 2x 15 min xylene, 2x 5 min 100% ethanol, 2x 5 min 96% ethanol, and 2x 70% ethanol. Slides were then washed 10 min with Tris‐buffered saline (TBS, 0.1 M, pH 7.6) including 0.3% Triton X‐100 (TBS‐T), 10 min TBS, and 10 min TBS‐T. Next, sections were blocked for one hour using 2% normal donkey serum (Sigma‐Aldrich, Zwijndrecht, The Netherlands, D9663) diluted in TBS‐T. Slides were then incubated overnight with a rabbit anti‐GABA polyclonal antibody (1:1000 diluted in TBS‐T; Sigma‐Aldrich, Zwijndrecht, The Netherlands, A2052). After rinsing unbound primary antibody in TBS (3x 10 min), slides were incubated with the far‐red secondary antibody Alexa‐Fluor 647 donkey anti‐rabbit IgG (1:100 diluted in TBS‐T; Invitrogen, Breda, The Netherlands) for two hours. Slides were then again washed in TBS (3x 10 min), after which slides were incubated with Hoechst (1:1000 in TBS) for 15 min in order to visualize nuclei. Last, slides were washed 3x 10 min in TBS, and then cover slipped with TBS/glycerol (80%/20%).