Alexa flour 532 conjugated phalloidin

Mice were deeply anaesthetized and trans-cardially perfused with 4% PFA. After removal of the spinal cord, spinal cord was post-fixed in 4% PFA overnight and subsequently incubated in 30% sucrose at 4 °C overnight. Thirty-five micrometers of thick free-floating sections were cut on a Leica 9000 s sliding microtome as described previously^{62 (link)} and collected in 0.1 M phosphate buffer (PB) pH 7.4. After incubation of the sections for 1 h with 4% normal goat or donkey serum and 0.3% Triton X-100 for blocking of nonspecific binding at room temperature, sections were incubated overnight at 4 °C with primary antibodies in blocking solution. After three times 10 min washing in 0.25% Triton X-100 in PB at room temperature, sections were incubated in with fluorescently labeled secondary antibodies, washed again and finally mounted with Mowiol/DABCO. The following primary antibodies were used: anti-NeuN (1:1000; Millipore, MAB377, clone A60), ChAT (1:1000; Millipore, MAB144P), anti-Ubiquitin (1:500; DAKO, Z0458), anti-Synaptophysin-1 (1:500; Synaptic Systems, 101 004). Alexa-647-, Alexa-488-, and Alexa-546-conjugated secondary antibodies were from Jackson Immuno-Research Laboratories. For visualization of F-actin Alexa Flour 532 conjugated Phalloidin (Invitrogen) was used.

For immunocytochemistry, cells grown on glass coverslips were fixed with buffered PFA for 20 min at RT, washed three times with PBS for 15 min, and blocked for 30 min with blocking buffer containing 10% donkey serum and 0.3% Triton X-100 in TBST (TBS-Tween). After three washes with TBST for 5 min at room temperature, cells were incubated with primary antibodies overnight at 4 °C, followed by three washes in TBST at RT for 15 min, and then incubated with the appropriate fluorophore-conjugated secondary antibodies for 1 h. Subsequently, cells were washes three times with TBST for 15 min. The coverslips were mounted on glass slides with Aqua Polymount. Cells were imaged using an Olympus Fluoview 1000i confocal microscope. The following primary antibodies were used: anti-phospho-Tau (Ser199/202) (1:500; Sigma-Aldrich, T6819), anti-Rab26 (1:500; Synaptic Systems, 269 011, clone 163E12), anti-Synaptophysin-1 (1:500; Synaptic Systems, 101 004). Alexa-647-, Alexa-488- and Alexa-546-conjugated secondary antibodies were purchased from Jackson Immuno-Research Laboratories. For visualization of F-actin Alexa Flour 532 conjugated Phalloidin- (Invitrogen) was used.