The other primary antibodies have all been used in the litera ture and assumed to be specific. F actin was visualized by incubating the cells with Alexa Fluor 488 conjugated Axitinib mw phalloidin. Occasionally, microglia were Inhibitors,Modulators,Libraries co labeled with FITC conjugated tomato lectin, which binds to N acetyl lactosamine residues on the microglia surface. In this case, after applying secondary antibody and washing, microglia were incu bated with tomato lectin and then processed as described above. Cell nuclei were labeled with 4,6 dia midino 2 phenylindole. After washing, cells on coverslips were mounted on glass slides with 50% gly cerol in PBS, VectaShield or Dako mounting medium. Dako mounting medium yielded more stable signals for longer imaging. For Orai1 and CaM staining, we used an antigen retrieval step after fixation.

Inhibitors,Modulators,Libraries Cover slips were microwaved on medium power for 3 min in citrate buffer, cooled in buffer and washed with PBS. Antigen re trieval is used to unmask epitopes that are obscured by crosslinking. Images were acquired with an Axioplan 2 wide field epifluorescence microscope equipped with an Axiocam HRm digital camera, and were analyzed with Axiovision 4. 6 software or ImageJ. Inhibitors,Modulators,Libraries For many images, we acquired Z stacks of high magnification epifluorescence images through the entire cell in 200 nm increments. These images were deconvolved using a theoretical point spread function and the constrained iterative maximum likelihood algo rithm in Axiovision 4. 7 software. Deconvolution reduces noise and distortions introduced during image acquisition.

It uses information about the optical system to calculate a point spread function of light above and below the plane of focus. Cell auto fluorescence and non specific staining were subtracted by using Inhibitors,Modulators,Libraries the same imaging and acquisi tion settings on cells exposed to a secondary antibody alone. When constructing Z stacks, the automated cor rection algorithm was used to compensate for fluores cence decay during repeated exposures. Migration, substrate degradation and invasion assays Live imaging Microglia were plated at 60,000 cells dish in 35 mm glass bottom culture dishes, and cultured for 2 days in MEM with 2% FBS. Cells were imaged for up to 1 hr using a Zeiss Axiovert 200M microscope, ORCA ER camera, Axiovision software and Neue LiveCell stage top incubator to maintain a temperature of 37 C and 5% CO2.

Scratch wound Microglia were seeded at 50,000 to 60,000 cells per 15 mm glass cover slip, and cultured in MEM Inhibitors,Modulators,Libraries with 2% FBS until approximately 80% confluent. The resulting monolayer of microglia was scratched with a sterile 200 ul pipette tip, with or without addition of a channel blocker. The cells were ZD6474 incubated for 24 hr, fixed and permeabilized as described above, and stained with FITC conjugated tomato lectin and DAPI. Five random fields along the border of the scratch were imaged at 10�� magnifica tion using a confocal microscope and saved.