Table 5.

Troubleshooting Guide for Visualization of dsRNA by Immunofluorescence

Problem	Possible Cause	Solution
Little to no sample remaining at the end of protocol	Loss of sample during wash steps	Increase centrifugation time, ensure pellet is visible after each wash
		Increase sample size (i.e. number of dissected intestines)
High background fluorescence	Antibody concentrations too high	Set up antibody titration to determine concentration that yields optimal signal- to-noise ratio
	Antibody incubation time too long	Decrease incubation time (i.e. 1 h at RT)
	Insufficient blocking	Make up fresh blocking buffer
	Insufficient removal of unbound secondary antibody	Increase number of washes following secondary antibody incubation