Alexa fluor 594 goat antimouse

Three to four-month old male mice were deeply anesthetized with chloral hydrate (400 mg/kg, i.p.) for transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate-buffered saline pH 7.4 (PBS). Their brains were dissected and stored in 4% paraformaldehyde for at least 24 h, and then washed with PBS and transferred to 30% sucrose in PBS. After the brains totally sank, 30 μm sections were cut with a freezing microtome.
For TN immunostaining, sections underwent heat-induced antigen retrieval by incubating them in 10mM NaCitrate for 30 min at a temperature of 70°C. After the sections cooled down to room temperature, they were washed with PBS three times, blocked with 3% BSA and 0.1% Triton X-100 in PBS for 1h, and then incubated with primary antibodies against TN [27] and tyrosine hydroxylase (TH; MilliporeSigma, Burlington, MA) overnight at 4°C. After three washes, the sections were incubated with Alexa Fluor 488 goat anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 594 goat anti-mouse (Jackson ImmunoResearch Laboratories) in 1.5% Normal Goat Serum (Vector Laboratories, Burlingame, CA) in PBS for 1h at room temperature. After three additional washes with PBS, the sections were mounted on slides, and coverslipped.
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