Table 1.

Troubleshooting Guide for ER-GCaMP6f Expression and Imaging

Problem	Possible Cause	Solution
ER-GCaMP6f is not expressed/Fluorescence is not observed in HEK-293 cells or astrocytes.	Plasmid concentration might have been low	Measure the concentration of plasmid and perform transfection with higher concentration of DNA
Too much background	Imaging media is not transparent	Replace the imaging media with L-15
Cell death is high	The plasmid DNA concentration might have been very high	Measure the concentration of plasmid and perform transfection with lower concentration of DNA
Transfection efficiency is low	Cells might have been overconfluent	Check the confluency of the cells and perform transfection when they are ~70% confluent
Cells are not focused on TIRF	It is probably because the dish is highly confluent, and cells are grown on top of each other	Check the cell confluency and make sure no cells are on top of each other before imaging
Animal is not recovered from anesthesia after surgery	The anesthesia dose might have been higher	Maintain ~2% isoflurane during the stereotactic surgery
No fluorescence is observed in slices	One reason might be not enough time for expression	Harvest slices after 3-4 weeks of ER-GCaMP6f expression and perform calcium imaging
Fluorescence decays very fast in cell culture or slice imaging	It might be because of high laser power or long exposure time	Decrease the laser power and/or exposure time
Movies/calcium images are oversaturated	The signal is too high	Decrease the laser power and/or exposure time