Table 1. Troubleshooting Table.

Step	Problem	Possible Reason	Solution
41	PDMS is not sealing to glass	PDMS not properly plasma treated	Remake channels
		Glass coverslip dirty	Boil new glass and remake channels
42	Channels will not fill with water after plasma sealing	Channels may be clogged with debris	Remake channels
		Channels not oxidized thus not hydrophilic	Remake channels
	Channels are leaking when filled with water	Channels not sealed with glass	Remake channels, check oxygen supply to plasma cleaner for leaks, pump down and re-fill oxygen line
		Surface of PDMS coated with contamination	Clean plasma cleaner and remake channels
46	Samples are not labeled or two-color labeled	Protein being labeled is not present or poor antibody	Try a different primary antibody
		Secondary antibodies are not tagging primary	A longer incubation may be required, try a different secondary antibody, use a fresh aliquot of secondary antibody
		Laser not exciting fluorophore	Try a different secondary with a different fluorophore, increase laser power, change camera settings, re-align setup or change filters
		Primary antibody not-reactive or expired	Use a fresh aliquot of primary, try different primary
	Individual regions of interest cannot be distinguished from background	The confluence of spots is too high	Re-load sample in channel with less sample
		Channels contaminated with fluorescence	Check buffer solutions for contamination
		Laser power too high	Adjust laser power to lowest possible setting
		Image not properly scaled	Adjust brightness and contrast in imaging software
		Samples mistakenly loaded in water	Reload samples in buffer
71	Dilution curve will not flatten out	Sample aggregation	Prepare new samples with sub-cellular compartments that have first been filtered with a size exclusion column or other similar technique.
		Excess free primary reacting with secondary antibody	Increase the amount of IgG beads added to sample, increase the amount of secondary antibody
107	Data is not reproducible	Samples not singly labeled or saturated	Re-check dilution curve
		Laser drifting	Check laser power over time, replace laser if needed
		Antibodies dissociating from sample	Image samples quickly after purification, try different antibody
		Incomplete labeling	Try longer incubation, optimize secondary antibody concentration
		Samples may be aggregating	Prepare new samples with sub-cellular compartments that have first been filtered with a size exclusion column or other similar technique.
		Improper background selection	Make sure there are no active ROIs near background selection area
		Camera or laser settings may have been changed between calibration and fully labeled samples	Use same settings