Donkey anti goat secondary antibody

Rat brain tissue or cell samples were lysed in RIPA buffer containing phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail as previously described. Lysates were centrifuged and collected. Equal amounts of total protein ranging from 15 to 30 μg total protein were separated by SDS-PAGE, and blotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The following antibodies were used: rabbit anti-cfos (1:1000, Abcam), rabbit anti-CX3CR1 (1:3000, Abcam), goat anti-FKN (1:200, Abcam), rabbit anti-BDNF (1:3000, Abcam), rabbit anti-GAPDH (1:3000, Servicebio), and goat anti-iba1 (1:500, Woko). Blots were developed with an HRP-labelled goat anti-rabbit antibody (1:5000, Servicebio) and an HRP-labelled donkey anti-goat secondary antibody (1:5000, Servicebio). Protein bands were visualized using a chemiluminescence system (ChemiDocTM XRS + , BioRad). Protein expression was semiquantitatively analysed with Image J software.

Western blot was performed to quantify the expression of SLC17A9 and ChAT. The methods were as described in the previous article. The membranes were incubated overnight at 4 °C with rabbit anti-SLC17A9 (1:1000; MBL), sleep anti-ChAT (1:1000; Abcam), and rabbit anti-actin (1:1000; Abcam; 1:1000; CST). Goat anti-rabbit secondary antibody (1:5000, BOSTER) and Donkey anti-goat secondary antibody (1:2000, Servicebio) in 5% non-fat milk were used to incubate for 1 h at room temperature.