AM1-44

Guidelines (The following is our recommended protocol. This protocol is for reference only and should be adapted to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Recommended concentration: 1-10 mM stock solution.

2. Working Solution Preparation
2.1 Diluent:
2.1.1 Depolarizing saline (50 mM KCl/75 mM NaCl substitution).
2.1.2 Tyrode's buffer containing 125 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 25 mM HEPES, 30 mM D-glucose, and 45 mM KCl.
2.1.3 Modified Tyrode's solution (150 mM NaCl, 4 mM KCl, 2 mM MgCl₂, 10 mM glucose, 10 mM HEPES, 2 mM CaCl₂; pH 7.4, approximately 310 mOsm).

2.2 Working concentration:
2.2.1 8 μM AM1-44 in depolarizing saline.
2.2.2 5 μM AM1-44 (in Tyrode's buffer containing 45 mM KCl, for cerebral cortical neurons; 5 μM was also used in DIV17-19 Wt cerebral cortical neuron experiments).
2.2.3 16 μM AM1-44 in modified Tyrode's solution.

2.3 Note: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Neuromuscular junction specimens^[1]:
3.1.1 Sample type: Neuromuscular junction (NMJ) specimens from the tibial nerve-flexor digitorum brevis and lumbrical muscles.
3.1.2 Incubation: Incubate with 8 μM AM1-44 in depolarizing saline.

3.2 Cerebral cortical neurons (adherent cells)^[2]:
3.2.1 Incubation: Incubate with 5 μM AM1-44 in Tyrode's buffer (containing 45 mM KCl) for 2 minutes.
3.2.2 Washing: Wash cells once after incubation; 5 minutes after washing, add 5 μM ADVASEP-7 to reduce background fluorescence.

3.3 Wt cerebral cortical neurons cultured in vitro for 17-19 days^[3]:
3.3.1 Sample type: Wt cerebral cortical neurons cultured in vitro for 17 to 19 days; cerebral cortical neurons were cultured from E16 to E18 Wt mice. AM1-44 is a nerve terminal probe and styryl dye used to load synaptic vesicles.
3.3.2 Incubation:
a. Incubate neurons with 5 μM AM1-44 for 5 minutes.
b. Incubate with 50 mM KCl for 5 minutes.
c. Incubate with 5 μM AM1-44 for 15 minutes.
d. Image AM1-44-loaded SVs under 5-minute baseline conditions.
e. Add 5 nM tPA or an equivalent volume of vehicle and image continuously for 60 minutes.
3.3.3 Washing:
a. After KCl incubation, perform several 1-minute washes with Ca²⁺/Mg²⁺-free HBSS.
b. After the second AM1-44 incubation, wash with Ca²⁺/Mg²⁺-free HBSS.
c. Incubate with 1 mM Advasep 7 for 10 minutes to wash off AM1-44 bound to exposed membranes.

3.4 Dissociated hippocampal cultures[4]:
3.4.1 Sample type: Dissociated hippocampal cultures prepared from 2- to 3-day-old Sprague-Dawley rat pups.
3.4.2 Use cultures after 15-25 days of in vitro culture.
3.4.3 Incubation: Incubate cultures with AM1-44 working solution in modified Tyrode's solution containing 1 μM tetrodotoxin (TTX) for 15 minutes.
3.4.4 Perform staining in the presence of 10 μM CNQX and 50 μM AP-5 to prevent recurrent activity.
3.4.5 Washing: After loading, wash in dye-free solution containing nominal Ca²⁺ for 10 minutes.

4. Controls
4.1 Vehicle control: Use an equivalent volume of vehicle as a control for 5 nM tPA during imaging. In release experiments, compare AM1-44 intensity in presynaptic terminals of vehicle controls.

5. Detection and Analysis
5.1 Instrument type: Fluorescence microscope, confocal imaging system, live-cell confocal microscope equipped with an oil-immersion ×40 objective, and confocal microscope; a cooled intensified digital CCD camera can be used for fluorescence imaging.

5.2 Result analysis:
5.21 AM1-44 fluorescence localizes to presynaptic motor nerve terminals, indicating functional synaptic vesicle recycling. In intact NMJs, colocalization with postsynaptic acetylcholine receptors stained with TRITC-α-BTX is expected; reduced or absent green fluorescence indicates NMJ denervation.
5.2.2 AM1-44 fluorescence intensity corresponds to dye loaded into presynaptic vesicles; decreased intensity indicates impaired presynaptic function. Fluorescence localizes to presynaptic vesicles. A second incubation with KCl induces release of vesicle-incorporated AM1-44, resulting in decreased fluorescence intensity.
5.2.3 AM1-44 fluorescence appears as puncta at presynaptic terminals and avoids dendrites and cell bodies. Compare AM1-44 puncta with syt1 lumen domain antibody labeling; colocalization indicates uptake into synaptic vesicles. Select isolated synaptic boutons (approximately 1 μm²) for analysis and avoid obvious synaptic clusters.