Cy2 or cy5 conjugated secondary antibodies

It was performed as described before.^{60 (link),63 (link)} Briefly, mice were anesthetized and perfused with PBS (pH 7.4) and then with 4% (w/v) paraformaldehyde solution in PBS followed by dissection of the brain from each mouse for IHC.^{64 (link),65 (link)} Samples were incubated in PBS containing 0.05% Tween 20 (PBST) and 10% sucrose for 3 h and then 30% sucrose overnight at 4°C. Brain was then embedded in O.C.T (Tissue Tech) at −80°C, and processed for conventional cryosectioning. Frozen sections (30 μm) were treated with cold ethanol (−20°C) followed by two rinses in PBS, blocking with 3% BSA in PBST and double labeling with two antibodies (Table S1). After three washes with PBST, sections were further incubated with Cy2 or Cy5 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). The samples were mounted and observed under the Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera. During Thio-S staining, after washing and incubation with secondary antibodies for 1 h, free-floating sections were stained with 0.002% Thio-S (Sigma) made up in TBS for 8 min. Sections were washed twice in 50% EtOH for 1 min and twice in TBS for 5 min before drying and mounting in Fluoromount (Sigma).