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Next, the silica particles were washed twice and wash buffers were removed by vacuum aspiration.
Contaminating enzymes and buffers were removed by purification with the Nucleospin PCR Clean-up Kit (Macherey-Nagel, Ger).
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Staining with anti-F4/80 antibody (diluted 1 : 400) conjugated with PE-Cy7 rat anti-mouse antibody was done for 30 minutes; antibody and staining buffer were removed by one time of washing in Hank's complete for ten minutes at 4°C and 500 g without brake and suspending in the same solution.
After the buffer was removed by filtration, the resin was shaken in a HEPES-buffered solution containing Zn(NO3 2 ⋅6 H2O (0.073 g, 0.24 mmol) at room temperature for 24 h.
The beads were then washed twice with 200 μl diluted BBB, and any excess of buffer was removed by aspiration with a pipette.
After 10 min on ice, the buffer was removed by aspiration.
Blocking buffer was removed by capillary action and the slides incubated over night at 4°C in either IP-10 goat polyclonal IgG (Santa Cruz G-15 sc-14641) or control goat IgG (Santa Cruz sc-2028) in 2% BSA.
Imidazole of the elution buffer was removed by dialysis.
At the end, the residual Offgel IEF buffer was removed by pipetting, and the electrode pads were removed.
Excess stop buffer was removed by washing the slide once in 50 mL of PBS for 2 minutes.
The assay buffer was removed by aspiration, and fluorescently-labeled detection antibody released by shaking in elution buffer (10 μl/well) for 5 minutes at 25°C.
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