HL-6: Artifical Drosophila hemolymph, with 5 mM L-glutamate added, and 2 mM Calcium.
Reagent
NA
NA
5 mM L-glutamate blocked muscle contractions. We used Molecular grade L-Glutamate (Sigma).
2 mM Calcium is close to physiological Calcium levels in natural larval hemolymph.
References: Macleod et al 2002 and Macleod 2004
Dextran, Alex Fluor 680; 10,000 MW, anionic, fixable
Reagent
Molecular Probes/Invitrogen
D34680
Use a small volume perfusion chamber to keep the total volume of dye low
Anti-HRP-CY5 conjugate (goat)
Reagent
Jackson ImmunoResearc
123-175-021
Dilute 2.0 mg into 1 ml ddH2O; aliquot into 4 microliter aliquots. Freeze at –20C. Dilute one aliquot into 100 microliters of HL-6
Alexa 647 antibody labeling kit
Reagent
Molecular Probes/Invitrogen
A10475
We prepared a total of 80 micro liters of conjugated primary antibody, and stored as 2 microliter aliquots. We diluted each aliquot into 100 microliter of HL-6 for labeling.
Custom Formulated Objective Oil, refractive index 1.3379
Brink, D., Gilbert, M., Auld, V. Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes. J. Vis. Exp. (27), e1154, doi:10.3791/1154 (2009).