One can do this by simply sucking out buffer 2 with a syringe.
AnnexinV and antibodies were filtered with a 0.1-µm filter (Millipore, Darmstadt, Germany), and PBS and AnnexinV-binding buffer with a 0.2-µm syringe filter (Sarstedt, Nümbrecht, Germany).
The gel wells were flushed with running buffer using a syringe just prior to sample loading.
Preparations were methanol fixed for 10 seconds and stained in 10% Improved R66 Giemsa (BDH) in phosphate buffer (pH 7.4), applied with a syringe containing a 0.45 µm pore size filter (Millex) to remove particulate matter.
Cerebellar slices, prepared as described above, were treated with 1 µM wortmannin or vehicle control for 10 min or with API-2 for 30 min in their recovery chamber before addition of DEA/NO for another 10 min. The slices were then transferred rapidly into 1 ml boiling 1.2× Laemmli SDS-PAGE sample buffer, homogenised by trituration with a syringe and 25-gauge needle, and further boiling.
The two channels of the flow chamber were continuously injected with appropriate buffers using a syringe pump (PHD2000; Harvard Apparatus, Holliston, Massachusetts) at a linear speed of 30 μm/s, which is approximately equal to the swimming speed of a healthy cell.
For Western analysis, regions with GFP expression (green) were micro-dissected out under a fluorescence microscope and homogenized with a syringe in lysis buffer followed by vortexing to prepare cell lysates.
Approximately 1 ml per 1 2 × 10 cells of hypotonic buffer was used to disrupt the cell membranes with a syringe and a narrow-gauge needle.
After ultracentrifugation, opaque liposome bands were collected with a syringe and diluted with detergent-free buffer to remove most of the sucrose and Triton X-100.
Vesicles were quickly thawed in a 37 °C waterbath, diluted to a protein concentration of 3.5 mg/ml with the appropriate resuspension buffer and revesiculated by 20 time aspiration through a 25G needle with a syringe.
