Table 1.

Troubleshooting guide for imaging of fixed and immunostained cells with DNA-PAINT-ERS

Problem	Possible Cause	Solution
Low conjugation efficiency for DS-DBCO or IS-dye	Loss of NHS-ester activity or improper pH for the reaction buffer	Use fresh reagents Confirm pH of the reaction buffer
Low recovery of DS-DBCO or IS-dye conjugates	Potential problem with DNA precipitation	Repeat DNA precipitation with DNA LoBind tubes Keep sample at −80°C degrees for longer Spin sample down at higher rpm or for prolonged time
Concentration of azide-tagged antibody is low	Problem with ultrafiltration	Passivate the spin filter with TWEEN 20 Check spin filter’s expiration date – the membranes used in spin filters occasionally fail
Low recovery of DS-conjugated antibody	Problem with ultrafiltration Failed conjugation	Check spin filter’s expiration date – the membranes used in spin filters occasionally fail Use fresh reagents Check pH of the reaction buffer
Unspecific staining	DBCO non-specifically bound to cellular targets Dirty coverglass	Quench DBCO with azide repeatedly Validate specificity of primary antibody Optimize blocking steps (% BSA, salmon DNA, signal enhancer) during low resolution imaging experiments Etch and clean coverglass
Low staining signal	Insufficient permeabilization Insufficient concentration of antibodies Too much blocking Solutions not fresh	Use triton instead of saponin (disadvantageous for membrane targets) Increase antibody concentration Block with lower % BSA Prepare fresh reagents
Nuclear staining	DBCO bound to nucleus	Add signal enhancer incubation step (see Basic protocol 2 step 13) Add salmon sperm DNA for additional blocking (see Basic protocol 2 step 15)
Uneven illumination after sample mounting	Bubbles in immersion oil Old oil smear on bottom of the coverglass from previous imaging session	Clean coverglass with isopropanol and objective lens with lens cleaning paper Remount sample with new immersion oil
Low signal to noise ratio during imaging	High IS concentration Wrong TIRF angle	Lower IS concentration to reduce background signal Adjust TIRF angle to select signal relevant to imaging plane of the target
Too slow/Fast DS/IS kinetics	Low/high room temperature EC concentration Orthogonal DS-IS pairs	Adjust EC concentration (5–15 %) Mix EC properly Monitor room temperature Check specific kinetics of DS-IS sequences Confirm the correct DS-IS pair is used
Unstable temperature in microscope room	Central climate control	Adjust EC concentration (5–15 %) Monitor room temperature and image during certain time windows
Focus drift	No focus stabilization Temperature changes during image acquisition Missing lid on the 8-well chambered coverglass	Introduce focus stabilization Equilibrate samples and imaging buffers at room temperature if previously stored in the fridge Place correct lid on top of the chambered coverglass to minimize evaporation of imaging solution
Lack of gold fiducials	Gold solution needs to be more concentrated or incubated for longer	Apply gold nanoparticles when sample is mounted on microscope, with laser illumination to assess proper amount of gold fiducials for a successful drift correction