Alexa 488 conjugated donkey anti mouse

Immunofluorescence staining was performed on frozen coronal sections of mouse brains or on cultured cells. The mouse brains were fixed with 4% paraformaldehyde. After fixation and concentration gradient dehydration, the brains were cut into 12-μm-thick sections. The cultured cells were fixed with 4% paraformaldehyde for 10 min. The brain sections and cell cover slips were washed three times with phosphate-buffered saline (PBS) and then incubated with primary antibodies overnight at 4°C in a humidified atmosphere. The following primary antibodies were used: mouse anti-NLRP3 (1:100; Adipogen, San Diego, California, USA), rabbit anti-NeuN (1:300; Millipore, Massachusetts, USA), goat anti–Iba-1 (1:200; Abcam, Cambridge, London, UK), and chicken anti–MAP-2 (1:200; Abcam, Cambridge, London, UK). Then, the samples were incubated with a mixture of Alexa-488–conjugated donkey anti-mouse (1:300; Abcam, Cambridge, London, UK), Alexa-594-conjugated donkey anti-rabbit (1:300; Abcam, Cambridge, London, UK), Alexa-594–conjugated donkey anti-chicken (1:300; Jackson ImmunoResearch, Pennsylvania, PA, USA), and Alexa-405–conjugated donkey (1:300; Jackson ImmunoResearch, Pennsylvania, PA, USA) anti-goat secondary antibodies for 2 h in the dark at room temperature. Finally, the sections were photographed using an Olympus BX51 (Tokyo, Japan) fluorescence microscope.