Brain sections were imaged using a Zeiss LSM700 laser scanning confocal microscope with 20X (numerical aperture (NA) 1.30) and 40X oil immersion (NA 1.25) objective lens (Zeiss). For all confocal images, stacks were acquired using 1 µm‐Z steps. Acquired images were adjusted for brightness and contrast using FIJI/ImageJ software.
CLARITY‐treated brains were mounted in a fructose‐based high refractive index solution (fHRI) prepared as follows: 70% fructose, 20% DMSO in 0.002 M PBS, 0.005% sodium azide. The refractive index of the solution was adjusted to 1.4571 using a refractometer (Krüss GmbH, Hamburg, Germany). In preparation for imaging, samples were incubated in 50% fHRI/50% PBST for 6 hr and finally incubated in 100% fHRI for at least 12 hr. For imaging, samples were mounted in 1% low melting point agarose and covered with fHRI. Whole‐mount brain fluorescence was captured using a Leica TCS SP8 laser scanning confocal microscope equipped with a Leica HC FLUOTAR L 25x/1.00 IMM motCorr objective. Fluorescence signal was detected by exciting the fluorophores with lasers at wavelength of 488 and 552 nm. Detection was performed by two internal photomultipliers (PMT).
CLARITY‐treated brains were mounted in a fructose‐based high refractive index solution (fHRI) prepared as follows: 70% fructose, 20% DMSO in 0.002 M PBS, 0.005% sodium azide. The refractive index of the solution was adjusted to 1.4571 using a refractometer (Krüss GmbH, Hamburg, Germany). In preparation for imaging, samples were incubated in 50% fHRI/50% PBST for 6 hr and finally incubated in 100% fHRI for at least 12 hr. For imaging, samples were mounted in 1% low melting point agarose and covered with fHRI. Whole‐mount brain fluorescence was captured using a Leica TCS SP8 laser scanning confocal microscope equipped with a Leica HC FLUOTAR L 25x/1.00 IMM motCorr objective. Fluorescence signal was detected by exciting the fluorophores with lasers at wavelength of 488 and 552 nm. Detection was performed by two internal photomultipliers (PMT).