2. Discard the washing buffer and rinse the membranes with 20 mL of blocking solution [Odyssey Blocking Buffer (LI-COR Bioscience, Lincoln NE, USA) and PBS 1× - (1 1)] for 15 minutes on a rocking platform.
Cell uptake was terminated at 5, 10, 15, 30, 60, 90, and 120 min by adding ice-cold Krebs buffer and rinsing the wells twice to wash away any unbound radioimmunoconjugates prior to lysing the cells with RIPA buffer.
After 45 min of incubation with shaking, the arrays were washed once with 200 μL of the buffer and then rinsed with water.
The surface was rinsed with buffer, and the sample dried.
The column was rinsed with washing buffer and the DNA fragments eluted with 50 μl of TE buffer.
Human macrophage cells were fixed for 1 hr with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, rinsed in the same buffer and the post-fixed with 2% osmium tetroxide in the same buffer for 1 hr at room temperature.
The slides were washed twice with fresh washing buffer and rinse with milliQ water.
The beads that contained the complete PRESSO product were first washed with 1.5 ml of TE buffer, and then rinsed with 200 μl of I- SceI buffer.
The reaction was stopped after 10 min incubation in the Stop Buffer and rinsed in 1xPBS.
Lysates were removed with sterile 3 mL syringes, and the filter units were each rinsed with 1 mL of lysis buffer and incubated for 15 min. The rinse buffer and lysates were pooled and then we performed the phenol-chloroform protocol as previously described [22].
