AM4-64

Guidelines (The following are recommended experimental protocols for guidance only and should be adjusted according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvent: Anhydrous DMSO.
1.2 Concentration Recommendation: 1-10 mM.

2. Working Solution Preparation
2.1 Diluents: Optional diluents include Murashige and Skoog (MS) liquid medium, Neurobasal medium, serum-free medium, PBS, Tyrode’s solution, and liquid Czapek-Dox medium.
2.2 Working Concentration: 5-24 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh immediately before use.

3. Staining Procedures
3.1 Adherent neuron-astrocyte co-culture system^[1]:
3.1.1 Sample Type: Adherent live neuron-astrocyte co-cultures; trypsin digestion is not required.
3.1.2 Incubation Conditions: Incubate with AM4-64 working solution for 5 min in a system containing 20 mM KCl.
3.2 Wild-type tobacco BY-2 cells^[2]:
3.2.1 Sample Type: Tobacco BY-2 cells
3.2.2 Incubate with 12 μM AM4-64 working solution on ice for 10 min.
3.2.3 Washing Step: Wash cells repeatedly with pre-chilled MS liquid medium to remove uninternalized dye.
3.3 Adherent human cortical neurons (HCNs)^[3]:
3.3.1 Sample Type: Adherently cultured HCNs; trypsin digestion is not required before staining.
3.3.2 Incubation Conditions: Co-incubate 5 μM AM4-64 working solution with 500 nM AβO plus 100 μM glutamate or 20 mM KCl for 5 min. For Tetrodotoxin pre-incubation experiments, pre-incubate neurons with 1 μM Tetrodotoxin for 20 min before adding AM4-64 (with or without AβO).
3.3.3 Washing Step: Rapidly wash the culture once with pre-chilled HBSS after incubation.
3.4 Adherent fungal hyphae^[4]:
3.4.1 Sample Type: Adherent fungal hyphae grown on the surface of cellophane placed on solid nutrient medium.
3.4.2 Incubation Conditions: Incubate with 6 μM, 12 μM, or 24 μM AM4-64 working solution at room temperature.
3.5 Adherent nerve growth factor (NGF)-differentiated PC12 cells^[5]:
3.5.1 Sample Type: Adherent NGF-differentiated PC12 cells.
3.5.2 Incubation Conditions:
3.5.2.1 First wash cells with Tyrode’s solution, then incubate in Tyrode’s solution containing 10 μM AM4-64 for at least 2 min.
3.5.2.2 Stimulate with depolarizing high-potassium Tyrode’s solution (70 mM KCl, 59 mM NaCl) containing 10 μM AM4-64 for 2 min.
3.5.2.3 Incubate in normal Tyrode’s solution containing 10 μM AM4-64 for at least 15 min (duration can be adjusted) to label slow endocytic processes.
3.5.2.4 Incubate in low-calcium Tyrode’s solution (0.2 mM CaCl₂, 5 mM MgCl₂) containing 0.5 mM SCAS for at least 5 min to quench uninternalized dye.
3.5.2.5 Fix cells with 3% paraformaldehyde and 0.5% glutaraldehyde solution for 1 h.
3.5.3 Washing Step: Wash cells with PBS after fixation.
3.6 Adherent Rhizoctonia solani hyphal cells^[6]:
3.6.1 Sample Type: Adherent R. solani hyphal cells grown on agar medium; directly use thin agar blocks with young hyphae.
3.6.2 Incubation Conditions: Incubate with 12 μM AM4-64 working solution for 8 min on ice in the dark.
3.6.3 Washing Step: Wash samples 3 times with pre-chilled liquid Czapek-Dox medium.

4. Control Setup
4.1 Human cortical neuron staining^[3]: Set up unstimulated control HCNs, glutamate-stimulated HCNs, and Tetrodotoxin-preincubated HCNs to verify vesicle cycle staining efficacy.
4.2 R. solani hyphal staining^[6]:
4.2.1 Inhibitor experiment control: Pre-incubate hyphae in nutrient medium without inhibitor first, then stain with AM4-64 following the standard protocol.
4.2.2 Plasma membrane permeability control: Incubate samples with 10 μg/mL propidium iodide in pre-chilled nutrient medium for 8 min, then observe under a microscope.

5. Detection and Analysis
5.1 Instrument Types: Fluorescence microscope, laser confocal scanning microscope, epifluorescence microscope.
5.2 Result Analysis:
5.2.1 Neuron-astrocyte co-culture system^[1]: AM4-64 fluorescence labels recycling synaptic vesicles as punctate signals; in normal co-culture systems, the density of AM4-64-positive punctate signals per 50 μm dendrite is higher than that in Down syndrome co-culture systems.
5.2.2 Tobacco BY-2 cells^[2]: Red fluorescence from internalized AM4-64 is detectable; over time, the dye first localizes to the plasma membrane, then to SCAMP1-labeled early endosomes, and subsequently to VSR-labeled prevacuolar compartments (PVCs); within 15 to 60 min, the co-localization percentage of AM4-64 with SCAMP1 increases from approximately 40% to approximately 90%, while that with VSR increases from 0% to approximately 80%.
5.2.3 Human cortical neurons^[3]: AM4-64 emits red fluorescence, labeling recycling synaptic vesicles on neuronal processes as fine punctate signals; glutamate stimulation increases the number of AM4-64-positive punctate signals, while TTX pre-incubation reduces this number; most AβO punctate signals are spatially separated from AM4-64-positive vesicles.
5.2.4 Fungal hyphae^[4]: AM4-64 labels primary endocytic vesicles, vacuolar membranes, and in some cases, mitochondria; evaluate endocytic activity based on primary vesicle concentration, initial vesicle appearance rate, and the time of vacuolar membrane signal emergence (15-25 min for S. hirsutum, 30-40 min for C. comatus, and 20-30 min for P. ostreatus).
5.2.5 NGF-differentiated PC12 cells^[5]: AM4-64 fluorescence appears as red punctate signals, localizing to neurite swellings, presynaptic-like structures at cell-cell contact sites, and perinuclear regions; approximately 32% of AM4-64 punctate signals overlap with synapsin punctate signals, among which approximately 40% of overlapping signals co-localize with Rab3a punctate signals; no stimulus-dependent fluorescence loss (fading) of AM4-64 punctate signals is observed upon high-potassium stimulation.
5.3.6 R. solani hyphae^[6]: The fluorescence localization process is divided into four endocytic stages: Stage 0 (plasma membrane only), Stage 1 (peri-wall primary endocytic vesicles), Stage 2 (larger endomembrane structures with weak membrane fluorescence), and Stage 3 (vacuoles of varying sizes with extremely weak membrane fluorescence); quantify the distribution of cells at each stage in the experimental and control groups at two time points: 0-15 min and 15-30 min.