Microchambers were prepared as detailed in Kumar et al [16 (link)]. In brief, a 22 X 22 mm, no. 1 (Fisherbrand, cat. # 12-548-B, Thermo Fisher Scientific, Waltham, MA) and a 50 x 24 mm coverslip (Fisherbrand, cat. # 12-545-F) were cleaned with laboratory detergent, rinsed with tap water and then de-ionized water before storage under ethanol. Double-sided tape 2 mm wide and 22 mm long was used to glue the two coverslips and form a channel lined with vacuum grease. (S2 Fig ).
Chambers were rinsed with two or more volumes of 20 mM Tris-acetate pH 7.9, 2 mM DTT, 100 mM potassium glutamate, 8 mM magnesium acetate and 0.5 mg/ml α-casein (TR buffer) by depositing a drop of buffer at one opening, allowing the liquid to enter by capillary action, and using a laboratory tissue to wick solution from the opposite opening and draw solution into/through the microchamber. 40 μl of 1:1000 solution of anti-HA antibody (12CA5; stock concentration of 1 mg/ml, Abcam, Cambridge, MA) diluted in TR buffer was added to the chamber and incubated at least 2 hours at room temperature or overnight at 4 degrees. Before use, the chambers were washed with 400 μl of TR buffer and incubated for 5 minutes.
Chambers were rinsed with two or more volumes of 20 mM Tris-acetate pH 7.9, 2 mM DTT, 100 mM potassium glutamate, 8 mM magnesium acetate and 0.5 mg/ml α-casein (TR buffer) by depositing a drop of buffer at one opening, allowing the liquid to enter by capillary action, and using a laboratory tissue to wick solution from the opposite opening and draw solution into/through the microchamber. 40 μl of 1:1000 solution of anti-HA antibody (12CA5; stock concentration of 1 mg/ml, Abcam, Cambridge, MA) diluted in TR buffer was added to the chamber and incubated at least 2 hours at room temperature or overnight at 4 degrees. Before use, the chambers were washed with 400 μl of TR buffer and incubated for 5 minutes.