Alexa 546 conjugated secondary antibodies

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.

Staining of NMJs was carried out as previously described^{54 (link)}. Briefly, mice were deeply anaesthetized and trans-cardially perfused with 4% paraformaldehyde (PFA). Subsequently, the muscles were dissected and post-fixed in 4% PFA for at least 2 hours. The tissue was washed in PBS-T (0.1% Tween-20) for 20 min at room temperature (RT) and incubated with ω-Bungarotoxin-Alexa-488 (Invitrogen) for 25 min at RT. The tissue was then incubated overnight at 4 °C with a blocking solution (2% BSA, 0.1% Tween-20 and 10% donkey serum), followed by incubation with the primary antibodies for 3 days at 4 °C. After washing with PBS at pH 7.4 (PAA Laboratories) thrice for 15 min, the appropriate secondary antibodies were applied for 1 h at RT. The tissue was washed again as above, and embedded in Aqua Polymount (Polysciences). The following primary antibodies were used: anti-NF-H (1:5000; Millipore, AB5539), anti-Synaptophysin-1 (1:500; Synaptic Systems, 101 004). Alexa-647 and Alexa-546-conjugated secondary antibodies were from Jackson Immuno-Research Laboratories.