Alexa fluor 488 donkey anti sheep igg

Rats were deeply anesthetized by pentobarbital (50 mg/kg, i.p.) and perfused transcardially with phosphate-buffered saline (PBS, composition in mM: NaCl 137, KCl 2.7, KH₂PO₄ 1.5, NaH₂PO₄ 8.1; pH 7.4) followed by 4% paraformaldehyde in PBS. Brains were removed and placed in fixative for two days and then stored in 25% sucrose solution for two days at 4℃, until frozen sections were cut for immunohistochemical staining. Brain sections were cut at 50 µm with a ROM-380 microtome (Yamato Kohki, Saitama, Japan). For immunohistochemical staining, the brain sections were incubated for 30 min at room temperature in a blocking solution (1% normal goat serum) and then were incubated for seven days at 4℃, in the primary antibody for muscarinic receptor (rabbit polyclonal anti-muscarinic M₁ receptor, 1:1,000; Millipore, Billerica, MA). After incubation, the brain sections were washed and incubated for 2 h at room temperature in the secondary antibody solution (Alexa Fluor 488 donkey anti-sheep IgG and Alexa Fluor 546 goat anti-rabbit IgG, 1:1,000; Invitrogen Corp., Carlsbad, CA). The brain sections were analyzed using an LSM510 Imaging System (Carl Zeiss GmbH, Jena, Germany).