Alexa 488 donkey anti-rabbit (1:400; Invitrogen), DyLight 488 donkey anti-chicken (1:400; Jackson ImmunoResearch, West Grove, PA) and/or DyLight 594 or 647 donkey anti-guinea pig (1:500; Jackson ImmunoResearch) secondary antibodies in 0.3% PBT for 2 hrs. Next, the sections were rinsed, mounted on slides with vectashield (with or without DAPI) (Vector Labs, Burlingame, CA), and coverslipped. Controls included processing the secondary antibodies alone to verify background staining, processing each primary with the secondary antibody to verify laser-specific excitation, examining for autofluorescence in an alternate laser channel with tissue lacking that laser-specific probe, and using sequential scanning. For subsequent fluorescent images, only the brightness and/or contrast levels were adjusted post-acquisition and were imposed across the entire image.
