Immunohistochemistry was done as described91 (link) except that the blocking step was overnight at 4°C. Primary antibodies: mouse anti-nc82 (DSHB, AB_2314866) 1:40, chicken anti-GFP (Abcam, ab13970) 1:1000, mouse anti-ChAT4B (DSHB, AB_528122), rabbit anti-GABA (Sigma, A2052). Secondary antibodies: Alexa-568 anti-mouse (Invitrogen) 1:400, Alexa-488 anti-chicken (Invitrogen) 1:400, Alexa-633 anti-mouse (Invitrogen) 1:400, Alexa-568 anti-rabbit (Invitrogen) 1:400.
Prolonged incubation (2-3 days at 4°C) with primary and secondary antibodies was required for homogeneous staining. Specimens were whole mounted in Vectashield (Vector Labs) on charged slides to avoid movement. Confocal stacks were acquired using a Zeiss 780 confocal microscope. Brains were imaged at 768 x 768 pixel resolution every 1 μm (0.46 x 0.46 x 1 μm) using an EC Plan-Neofluar 40x/1.30 oil objective and 0.6 zoom factor. All images were acquired at 16-bit color depth. Maximum projections of z stacks were made in Fiji.89 (link)
Prolonged incubation (2-3 days at 4°C) with primary and secondary antibodies was required for homogeneous staining. Specimens were whole mounted in Vectashield (Vector Labs) on charged slides to avoid movement. Confocal stacks were acquired using a Zeiss 780 confocal microscope. Brains were imaged at 768 x 768 pixel resolution every 1 μm (0.46 x 0.46 x 1 μm) using an EC Plan-Neofluar 40x/1.30 oil objective and 0.6 zoom factor. All images were acquired at 16-bit color depth. Maximum projections of z stacks were made in Fiji.89 (link)
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