Alexa fluor 488 conjugated donkey anti rat

Mice were anesthetized with 1% pentobarbital (i.p.) and sacrificed by transcardial perfusion while deeply anesthetized, as described above. The post-fixed brains were sliced to 30 μm thickness using a cryotome (Leica, CM1950, German). The collected slices were first incubated for 15 min in immunostaining permeabilization buffer containing Triton X-100 (P0096, Beyotime, China), followed by 15 min in immunostaining blocking buffer (P0260, Beyotime, China), and finally in buffer containing primary antibodies, namely mouse anti-GFAP (1:200; 3670, CST, USA), rabbit anti-AQP4 (1:50; 16473-1-AP, Proteintech, China), rabbit anti-AQP4 (1:200; A13168, ABclonal, China), and rat anti-CD31 (1:200; 550274, BD Biosciences, USA) overnight at 4 °C. After rinsing three times in PBS, slices were incubated with corresponding secondary antibodies, including Alexa Fluor 647-conjugated donkey anti-mouse (1:400; Abcam, USA), Cy3-conjugated goat anti-rabbit (1:400; Jackson Immunoresearch, USA), and Alexa Fluor 488-conjugated donkey anti-rat (1:400; Jackson Immunoresearch, USA) for one hour at room temperature. After dehydration, slide mounting, and clearing the immunofluorescent images were captured by laser scanning confocal microscopy (Olympus, FV1000, Japan) and analyzed using Fiji software by a blinded investigator.

Tissue sections were incubated overnight at 4 °C with the following anti-mouse antibodies: Ly-6G/Ly-6C (Gr-1)-APC/Cy7 (Biolegend, 108424), Meca32 (BD, 550563). For Meca32 staining an Alexa Fluor 488-conjugated Donkey Anti-Rat (Jackson ImmunoResearch laboratories; 712-546-153) was applied for 1 h at RT. Sections were washed with acidified water, and mounted with DAPI Fluoromount-G (Southern Biotech; 0100-20). Images were obtained at ×20 or ×40 magnification using Leica DM4000B microscope and digital camera (Leica DFC 360FX). Brightness and contrast were adjusted equally in all images presented.

The following primary antibodies were used in this study: rat monoclonal anti-α-tubulin (MCA77G; AbDSerotec), mouse monoclonal anti-GM130 (610823; BD Transduction), rabbit polyclonal anti-pericentrin (432-C; Covance PRB), rabbit polyclonal antibodies against either pan-N-WASP or phospho-N-WASP (4848; Cell Signalling, AB23394; Abcam), rabbit polyclonal anti-PKCζ C-20 (SC-216; Santa Cruz Biotech), mouse anti-EEA1 (610457; BD biosciences), and HRP coupled anti-GFP (ab6663; Abcam). As secondary antibodies, we used standard antibodies from Jackson ImmunoResearch: Cy5 conjugated donkey anti-mouse, Alexa Fluor 488 conjugated donkey anti-rat, TRITC conjugated donkey anti-rabbit, as well as HRP coupled donkey anti-mouse, anti-rabbit, and anti-goat. DAPI in ProLong Gold Antifade Reagent (Life Tech) was used to visualize nuclei. To suppress lipid modification of CDC42 isoforms, cells were treated overnight in 120 µM 2BP (to suppress palmitoylation; Sigma-Aldrich) or 20 µM GGTI298 (to suppress geranyl-geranylation and palmitoylation; Tocris).