As described previously (18 (link)), animals were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde in phosphate-buffered saline, pH 7.4. The brain was extracted and postfixed in 4% paraformaldehyde for at least 12 h before transferring to a cold 30% sucrose solution for >48 h. Brains were then cryosectioned coronally at 40-μm thick on a sliding microtome.
Brain sections were mounted on Superfrost Plus (Fisher Scientific) slides and coverslipped with DPX or Vectashield with DAPI (Vector Laboratories). A microscope with a 100 W mercury lamp with fluorescence optics (Leica Microsystems) was used to image the sections and photos were taken with a Retiga 2000 monochrome CCD camera and Q Capture Pro software (Qimaging). Leica TX2 filter cubes (excitation 560 nm, emission 645 nm, dichroic 595 nm) were used to visualize Fluoro-Ruby and mCherry fluorescence, L5 filter cubes (excitation 480 nm, emission 527 nm, dichroic 505 nm) were used to visualize GFP and eYPF fluorescence. Q Capture Pro software and FIJI (NIH) were used to overlay images and adjust brightness and contrast. High-resolution photomicrographs of input cells were captured with LAS AF Leica software on a Leica SP5 Tandem Scanner Spectral 2-phtoon confocal microscope.
Brain sections were mounted on Superfrost Plus (Fisher Scientific) slides and coverslipped with DPX or Vectashield with DAPI (Vector Laboratories). A microscope with a 100 W mercury lamp with fluorescence optics (Leica Microsystems) was used to image the sections and photos were taken with a Retiga 2000 monochrome CCD camera and Q Capture Pro software (Qimaging). Leica TX2 filter cubes (excitation 560 nm, emission 645 nm, dichroic 595 nm) were used to visualize Fluoro-Ruby and mCherry fluorescence, L5 filter cubes (excitation 480 nm, emission 527 nm, dichroic 505 nm) were used to visualize GFP and eYPF fluorescence. Q Capture Pro software and FIJI (NIH) were used to overlay images and adjust brightness and contrast. High-resolution photomicrographs of input cells were captured with LAS AF Leica software on a Leica SP5 Tandem Scanner Spectral 2-phtoon confocal microscope.
