Alexa 488 or alexa 594 conjugated secondary antibody

Brain sections were rinsed twice for 10 min each in PBS at pH 7.4, and then incubated in nonspecific binding blocking solution (0.1 M PBS containing 0.2% Tween-20 and 15% normal goat serum; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 1 h at room temperature (RT). The sections were then incubated overnight at 4 °C with primary antibodies (see Table 3), diluted in blocking buffer. Negative primary controls included tissues treated with blocking solution but without the addition of primary antibodies. The sections were then washed in PBS and incubated with Alexa-488- or Alexa-594-conjugated secondary antibodies (ThermoFisher Scientific, Waltham, MA, USA), diluted 1:500 in blocking buffer, for 2 h, at RT. The tissues were then washed three times with PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min, and coverslipped with Vectashield antifade mounting medium (Vector Labs, Burlingame, CA, USA). Immunostained images were captured with a Leica DM6 B upright microscope (Leica Microsystems, Northbrook, IL, USA) and Leica Application Suite X (LAS X) software version 3.5.5. For each tissue section, area/pixel analysis software (Pixcavator 4) was used to quantify the number of pixels inside the outer boundary of each cell body; this aided quantification of the density of immunofluorescence cell markers relative to background.