0.25na objective

Mice were anesthetized and perfused intracardially with 0.01 M PBS followed by 4% PFA. Brains were stored in 4% PFA for 12–18 hours at 4°C before being washed three times in 0.01 M PBS. Slices were cut on a VT-1000S vibratome (Leica) at 75 μm thickness and placed on gel-coated glass slides. ProLong Gold anti-fade reagent with DAPI (Invitrogen) or VectaShield with DAPI (Vector Labs) was applied to the surface of the slices, which were then covered with a glass coverslip. Fluorescent images were taken on an Olympus VS120 microscope, using a 10X 0.25NA objective (Olympus) or on a Leica TCS SP8 confocal microscope, using either a 10X 0.4NA or 20X 0.75NA objective (Olympus).

Mice were anesthetized and perfused intracardially with 0.01 M PBS followed by 4% PFA. Brains were stored in 4% PFA for 12 – 18 hours at 4°C before being washed three times in 0.01 M PBS. Slices were cut on a VT-1000S vibratome (Leica) at 70 μm thickness, or 40 μm for immunostaining, and were either mounted directly onto gel-coated glass slides or processed for immunolabeling. For tdTomato immunolabeling, slices were incubated for 1 hour in blocking solution (1% bovine serum albumin and 0.2% Triton-X in 0.01 M PBS), primary antibody was applied overnight (rabbit anti-RFP (Rockland) at 1:1000 in blocking solution). Slices were then washed in PBS and incubated with secondary antibody (goat anti-rabbit 594 (abcam) at 1:400), in blocking solution) for 1.5 hours. Slices were coverslipped using ProLong Gold anti-fade reagent with DAPI (Invitrogen) or VectaShield with DAPI (Vector Labs). Fluorescent images were taken on an Olympus VS120 microscope, using a 10X 0.25NA objective (Olympus) or a Leica TCS SP8 confocal microscope, using a 20X 0.75NA and 40X 1.3NA objective (Olympus). All images were processed using NIH ImageJ.