508 The Journal of Cell Biology | Volume 159, Number 3, 2002 detach the neurite. Large numbers of neurons successfully manipulated were lost as a result of attempted needle removal. Cultures that were incu- bated after experimental manipulations were maintained in glia-condi- tioned CO2-independent medium containing horse serum and supple- ments as described above or in this same medium with an overlay of rat glia on a coverslip (Goslin and Banker, 1991). Immunofluorescent analyses Experimental cells successfully removed from the needle were marked with a circle made on the bottom of the dish with a diamond-tipped micro- scope “objective.” Immunofluorescent staining for dephospho-tau was performed by a method similar to that of Ruthel and Hollenbeck (2000). Medium was carefully removed, and the cells were fixed in a freshly pre- pared solution of 4% paraformaldehyde and 4% sucrose for 20 min. After two rinses in PBS, the cells were permeabilized by a 10-min incubation in 0.2% Triton X-100 in PBS. Cell were then incubated in 3% goat serum and 2% BSA in PBS for 30 min and then in an antibody specific for dephospho- tau (tau-1, 1:200; Boehringer) for 2 h at 37 C. After three rinses with PBS, cells were incubated in Alexa-488–conjugated secondary antibody (1:1,000; Molecular Probes) for 1 h at room temperature. After three more rinses in PBS, coverslips were mounted over the appropriate region of the tissue culture dish with Aqua Polymount (Polysciences Inc.). Immunofluo- rescent staining for L1 was performed by a similar procedure except that the permeabilization step was omitted and the primary anti-L1 antibody was a gift from Dr. Vance Lemmon (Case Western Reserve University, Cleveland, OH). This was diluted 1:10,000 in 2% horse serum in PBS and incubated with the sample overnight in the refrigerator as recommended by Dr. Lemmon. Immunofluorescent staining for microtubules was performed by a method similar to that of Thompson et al. (1984). Cells were lysed and sta- bilized for 3 min at 37 in 0.5% Triton X-100 in the microtubule-stabilizing buffer described by Thompson et al. (1984). The cells were then fixed for 20 min with freshly prepared 4% paraformaldehyde in microtubule-stabi- lizing buffer. After rinsing with PBS, cells were blocked with 3% goat se- rum and 2% BSA in PBS for 30 min. Cells were then reacted with a pri- mary antibody against -tubulin (1111876; Boehringer) for 1 h and washed three times with PBS. The fluorescent secondary antibody was goat anti–mouse IgG AlexaFluor 488 (A11001; Molecular Probes). After rinsing three times in PBS, the cells were mounted as above. We are grateful to Vance Lemmon for his kind gift of anti-L1 antibody and for his suggestions as to dilution and protocol with this antibody. 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