View source for Micropatterning Proteins

This protocol is based on IgG micropatterning methods as published by Freeman ''et al''. <ref>Freeman et al., (2016) “Integrins Form an Expanding Diffusional Barrier That Coordinates Phagocytosis.” Cell. V164#1, pp 128-40. http://linkinghub.elsevier.com/retrieve/pii/S0092867415015676</ref>It may need to be optimized for other proteins. 

==== Preparation of the Microprinting Stamp. ====
This is optimized for a PDMS based stamp and may have to be modified for stamps made of different materials. It is best to perform this procedure after each use of the stamp; the stamp can then be briefly rinsed with 70% ethanol before use.

# Press the stamp gently against a piece of scotch take, and then pull the tape away.
# Soak stamp in diluted laboratory glassware cleaner, 20 min.
# Rinse 5 times with dH<sub>2</sub>0.
# Rinse with 70% ethanol.
# Allow to air dry.

==== Patterning a coverslip. ====
''Acid washed &/or plasma cleaned coverslips should be used for this procedure.''

# Dilute IgG<sup>1</sup> to 1 mg/mL, preparing ~20 μL per coverslip to be patterned.
# Tape a piece of parafilm to the bench.
# Place a 20 μL drop of the IgG solution on the parafilm.
# Place the stamp, pattern-side down, on top of the drop.
# Incubate at room temperature for 30 minutes.
# Lift the stamp and gently dry under a slow flow of air.
# Place the stamp on a cleaned coverslip and place a light weight (e.g. 1.5 mL tube filled with water) on top of the stamp.
# After 10-20 seconds, remove the stamp. If preparing other coverslips, repeat steps 3-8 for the additional coverslips.
# Passivate the coverslip by placing 0.1 mg/Ml of poly(L-lysine)-graft-poly(ethylene glycol) [PLL-g-PEG] in PBS for 30 min at room temperature<sup>2</sup>.
# Rinse once with water and store until needed.
# Clean the stamp and store, pattern-side-up, in a sealed container.

<sup>1.</sup> Unlabeled or fluorescently-labeled IgG can be used. If using unlabeled IgG, a fluorescent secondary antibody can be used to detect the IgG spots after the experiment is completed.

<sup>2.</sup> Passivation prevents proteins and cells from adhering directly to the glass. Depending on the substrate, this step may or maynot be needed.