Alexa fluor 488 donkey anti mouse igg a21202

The rats were deeply anesthetised with an overdose of chloral hydrate (10%, 0.3 ml/100 g) and transcardially perfused with 200 ml of 0.01 M PBS (pH 7.4), followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The L4–L6 spinal cord segments with the spinal dorsal horn (SDH) were harvested and dehydrated in 30% sucrose at 4°C. Transverse spinal sections (30-μm-thick) were then cut using a cryostat (CM3050S; Leica, Wetzlar, Germany). For double immunofluorescence, the sections were incubated with rabbit anti-c-Fos (1:500; ab209794; Abcam, Cambridge, MA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:400; #3670; Cell Signaling Technology, Danvers, MA, USA) and goat anti-IBA-1 (1:500; MABN92; Millipore, Billerica, MA, USA) antibodies, followed by FITC-conjugated secondary antibodies [Alexa Fluor 594 donkey anti-rabbit IgG (A21207), Alexa Fluor 488 donkey anti-mouse IgG (A21202) and Alexa Fluor 488 donkey anti-goat IgG (A11055); 1:500; all from Invitrogen, Carlsbad, CA, USA] for 2 h at room temperature. The stained sections were observed and captured under a confocal laserscanning microscope (FV1000; Olympus, Tokyo, Japan).