For western blotting, total cell proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a 4 12% bis/tris acrylamide (Novex NuPAGE, Invitrogen) gradient gel using the Nupage system with Mops SDS buffer under reducing conditions and transferred for 3 h at 60 V onto a nitrocellulose membrane.
Protein (50 μg) was separated on 4 12% Bis/Tris NuPage gels in MES buffer and transferred to nitrocellulose for 60 90 minutes at 30 V.
Finally, the cells were washed twice, resuspended in staining buffer, and transferred to FACS tubes for analysis.
Finally cells were resuspended in 0.5 ml of the same buffer and transferred to FACS tube for flow cytometry analysis.
Cells were pelleted, washed twice, resuspended in ∼300 µl of FACS staining buffer and transferred to flow cytometry tubes for cytometric analysis [33].
The cells were washed with Tyrode buffer, resuspended in 2.5 ml Tyrode buffer and transferred to a SPEX fluorimeter programmed for excitation at two wavelengths (340 and 380 nm) with an emission at 505 nm, under constant stirring at 37°C.
Tissues were washed with HEPES buffer and transferred to 0.1 M sodium cacodylate buffer (pH 7.3), postfixed in 1% OsO4 in 0.1 M cacodylate buffer (pH 7.3) for 1 h and rinsed in 0.1 M sodium cacodylate buffer (pH 7.3).
Of this 500 µl sample 200 µl was transferred into a HPLC reaction vial and diluted 1 1 with 200 µl 0.05 M pH 6.8 phosphate buffer solution and transferred into the HPLC machine for analysis.
The sampled larvae were euthanized with an overdose of 4 g/L buffered tricaine and transferred to different buffers and fixatives for subsequent analyses.
The sections were then incubated in primary antibody diluting buffer (Golden Bridge International, Mukilteo, WA, USA) and then transferred for overnight incubation with IbaI antibody (WAKO, Osaka, Japan), CD39 (R&D, Minneapolis, MN, USA), and CD11b (Serotec, Raleigh, NC, USA) at 4 °C.
