ATK Brain Atlas Spatial Validation Images

Attached are maximum z-projected images of each of our brain samples for spatial validation. These are compressed tiffs with all channels merged due to the large size of each image (.ims or .lif) file. Thus, raw files are available upon request. Each sample was imaged using a Leica Stellaris 5 confocal microscope equipped with a DMI8 inverted microscope stand. The objectives used to both locate and image tissue were 10X dry objective lens (10x/0.4 HC PL APO CS2 506424) and a 63x oil-immersion lens (63x/1.4 HC PL APO OIL CS2 506350) lubricated with Leica Microsystems Type F immersion oil (11513859). Confocal z-stacks using a 0.3mm step size were taken for each. Cell-type 4-plex images were taken with a 1024 x 1024 resolution at 600Hz (8000Hz for the mifepristone experiment), and DE gene sets were imaged with a 512 x 512 resolution using the resonant scanner (8000Hz) built into the Stellaris system. Laser intensities were 10 for all channels with an automatic digital gain of 2.5%. The laser mode was chosen based on the fluorophores’ excitation and emission spectra: DAPI for Channel 1, FITC for TSA Vivid fluorophore 520 (Channel 2), Cy3 for TSA Vivid fluorophore 570 (Channel 3), Texas Red for Opal 620 dye (Channel 4), and Cy5.5 for Opal 690 dye (Channel 5). Each image consisted of one hemisphere of the brain slice containing the optic tectum which was anatomically guided by the coronal brain atlas in D’Angelo (2013).
For 4-plex cell-type images the channel information is as follows: Channel 2 is apoeb (yellow), Channel 3 is mpz (green), s100b (magenta), and map2 (red). For our first DE gene set, custom-designed probes were hybridized to zinc finger NFX1-type containing 1 (znfx1; green), helicase with zinc finger domain 2 (helz2; yellow), and interferon induced with helicase C domain 1 (ifih1; red). The second DE gene set’s probes were hybridized to optineurin (optn; green), interferon regulatory factor 3 (irf3; yellow), and signal transducer and activator of transcription 1a (stat1a; red). Both DE gene sets used TSA Vivid Fluorophore 570 for C1, Opal 620 for C2, and Opal 690 for C3.