Alexa 488 conjugated goat anti mouse igg

Immunohistofluorescence assay was performed according to the method described before [19 (link)]. Anti-α-SMA antibody and anti-F4/80 (Abcam, UK) were used. All of these antibodies were used at a dilution of 1/100. Alexa 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) and Alexa 568-conjugated goat anti-rabbit IgG (Invitrogen) were used as secondary antibodies.

Sections were deparaffinized and rehydrated. Antigen retrieval was done by heating. In order to reduce autofluorescence, Sudan black B treatment was performed. Sections were immersed in 5 % skim milk in TBS. After incubation with each of the primary antibodies at 4 °C overnight, the sections were washed with TBS. The sections were then incubated with corresponding secondary antibodies at 37 °C for 1 h, mounted with Vectashield (H-1500, Vector Laboratories, Burlingame, CA), and examined under a Leica DMI 3000B fluorescence microscope (Leica Microsystems, Tokyo, Japan) or a Carl Zeiss LSM700 Confocal Laser Scanning Microscopy (Carl Zeiss, Tokyo, Japan). Primary antibodies used were as follows: mouse anti-hyperphosphorylated-tau Ser202/Thr205 (clone AT8, 1:100, Thermo Scientific), rabbit anti-MAP2 (1:1000, Millipore), rabbit anti-GFAP (1:400, Dako), rabbit anti-Olig2 (1:200, Millipore), mouse anti-RAB9 (clone Mab9, 1:100, LSBio, Seattle, WA), and rabbit anti-Aβ42 (1:100, IBL). Secondary antibodies used were as follows: ALEXA594-conjugated goat anti-mouse IgG (1:100, Invitrogen, Eugene, OR), ALEXA488-conjugated goat anti-rabbit IgG (1:100, Life Technologies, Eugene, OR), ALEXA594-conjugated goat anti-rabbit IgG (1:100, Life Technologies), and ALEXA488-conjugated goat anti-mouse IgG (1:100, Invitrogen).