Phalloidin cf568 conjugate

Plasticware was obtained from Costar (Merck, Darmstadt, Germany) and Sarstedt (Nümbrecht, Germany). Materials for real-time quantitative PCR were obtained from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA). Organic solvents for TLC, paraformaldehyde, glutaraldehyde, and Hoechst 33342, were obtained from Sigma-Aldrich (St. Louis, MO). Phalloidin-CF568 conjugate was obtained from Biotium (Fremont, CA). Bodipy 493/503 was obtained from Thermo Fisher Scientific.
Lipids used for TLC were purchased from two sources: 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (#42773), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine (#01991), and 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-L-serine sodium salt (#51581) were obtained from Sigma-Aldrich (St. Louis, MO). Hexadecanoic acid (palmitic, #N-16-A), octadecanoic acid (stearic, #N-18-A), 9-hexadecenoic acid (palmitoleic, #U-40-A), 9-octadecenoic acid (oleic, #U-46-A), 7-octadecenoic acid (vaccenic, #U-48A), 9-12 octadecadienoic acid (linoleic, #U-59-A), trihexadecenoin (palmitolein, #T-215), 13-docosenoic acid (erucic, #U-79-A), dipalmitolein (#D-216), trioctadecenoin (#T-235), diolein (#D-236), diolein 1-3 Isomer (#D-237), monoolein (#M-239), cholesteryl palmitate (#CH-815), cholesteryl palmitoleate (#CH-826), and cholesteryl oleate (#CH-828) were obtained from Nu-Chek Prep Inc. (Elysian, MN).

Neurons were cultured on 4 well slides. At 1DIV, neurons were transduced with GFP lentivirus. On 6DIV, neurons were treated with nocodazole and fixed for 10 minutes in 2%PFA. Samples were washed 3x 5 minutes and subsequently stained with Phalloidin, CF568 Conjugate (Biotium) for 30 minutes. Neurons stained for Tuj1 (T2200, Sigma) 1:1000. Neurons were washed 2x 5 minutes and mounted using VECTASHIELD® HardSet™ Antifade Mounting Medium, Liquid (Vector Laboratories).
To investigate microtubule integrity, neurons were briefly washed (1 minute) in cytoskeletal extraction buffer (1 mM Magnesium Chloride, 1mM EGTA, 0.1M 1,4-Piperazinediethanesulfonic acid (PIPES), 0.5% Triton X-100), fixed for 10 minutes in 1% glutaraldehyde (Electron Microscopy Sciences 16019), reduced in freshly prepared 0.1% Sodium Borohydride (Sigma 452882), washed 2x5 in PBS, and stained using the Immunostaining media kit (Active Motif, cat. no. 15251). Neurons were stained with 1:500 detyrosinated tubulin antibody (Sigma AB3201) and neurofilament 200 (Sigma N4142).