Alexa fluor 594 conjugated donkey anti mouse secondary antibody

CLARITY-processed brains were cut using a rat brain slicer (Adult Rat Brain Slicer Matrix BSRAS002-1, Pittsburgh, PA, USA) to 2 mm thick coronal slices. The brain slices were then washed with 0.1 M PBS, pH 7.4, for 24 hours. The specimens were incubated with primary antibody diluted in 0.1 M PBS and 0.3% m/v Triton-X with the addition of 0.01% m/v sodium azide for 7 days on an orbital shaker at room temperature (RT). The primary antibody was a rabbit polyclonal anti-Iba-1 (Wako, Osaka, Japan, 1:100) for rat brain. Immunohistochemistry of humans brain was carried out using 100 fold dilution of rabbit polyclonal anti-tyrosine hydroxylase antibody (TH, Millipore, Temecula, CA, USA, 1:100) and mouse monoclonal anti-parvalbumin antibody (PV, Sigma, St. Louis, MO, USA, 1:100). After incubation with the primary antibody, the sections were washed with 0.1 M PBS for 24 hours. Tissue slices were incubated with AlexaFluor 488 conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, Baltimore, MD, USA) and AlexaFluor 594 conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch, Baltimore, MD, USA).. The secondary antibodies were 100 fold diluted in 0.1 M PBS and 0.3% m/v Triton-X and incubated on an orbital shaker at RT for 7 days. The tissue was then washed with 0.1 M PBS for 24 hours at RT to remove excess unbonded antibody.