Table 5:

Troubleshooting

Problem	Possible Cause	Solution
Inflated lungs during perfusion, liquid coming from mouth or nose	The needle might have inserted into the right atrium or right ventricle instead of the left ventricle	Remove the needle and insert it into the left ventricle
Fixation tremor is not observed during paraformaldehyde perfusion/fixation	The needle might have inserted into the wrong place	Re-insert the needle into the left ventricle
Blood vessels can be seen in the perfused brain	Perfusion was unsuccessful	Perform perfusion by inserting a syringe needle in the left ventricle and make a cut in the right atrium Increase the volume of solutions used or the timing of the perfusion process
The brain is too soft in clearing solution	The brain was not fixed properly	Perform post-extraction fixation for a longer time 48-72 hours in 4 % paraformaldehyde (in PBS)
Brain tissue becomes unclear/opaque after storing in PBS for a long time	The tissue might have absorbed salt	Perform TDE clearing for 24-48 hours
Fluorescence is too low	Antibody concentration might not have been enough, Exposure duration was not enough	Use a higher concentration of antibody and incubate for a longer period
Vasculature/blood vessels are observed during astrocyte imaging	Perfusion was unsuccessful	Perform perfusion by inserting a syringe needle in the left ventricle and make a cut in the right atrium Perform perfusion with a higher volume of PBS
The fluorescence signal is not obtained from the inner part of the tissue	Antibody might have difficulty in reaching inner sections of the brain	Perform longer incubation and use 1:50-1:100 concentration of antibodies Use polyclonal booster antibodies (such as anti-GFP alexa 488 which will enhance GFP fluorescence at the same fluorescence channel) If possible, perform brain sectioning (hemibrains) or slicing so that antibodies can reach to inner parts easily