Table 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
26	Tissue is leaky/dye is spreading	Poor perfusion/perfusate	Ensure that the perfusate does not contain sodium chloride, is not heated above 53° C, is of pH between 7.2 and 7.4 and is prepared the day of perfusion
		Too long time elapses between opening the chest cavity and start of perfusion	Practice perfusions until there is minimal delay between opening the chest and initiating the perfusion, with no more than a few seconds elapse between impaling the needle in the left ventricle, initiating flow, and opening the right atrium.
		Postfixation too short for specific tissue	Increase the postfixation time
28	Dye enters the soma but doesn’t seem to spread into the distal dendrites	Postfixation too long for specific tissue	Decrease the postfixation time
29	Dye doesn’t	Tip is clogged (for LY, tip will appear red)	With tip away from slice but inside PBS, apply opposite current (i.e. positive current) of 10–40 nA for a few seconds. If tip still clogged, switch to a new glass micropipette
		Micropipette tip too small	Adjust puller to give pipettes with slightly less resistance
		Tip is resting on membrane or nucleus	Tap manipulator to unclog tip; also try very minute readjustments of pipette
29	Current injector is beeping and no dye is coming out	The current circuit is broken	Check entire current path; if platinum is coated, remove coat with a razor blade; if the platinum wire has kinks in it, replace it; for air objective setups, ensure that there is enough liquid to create a current path between micropipette and ground
26–30	Dendrites appear beaded	Too long time elapses between opening of chest cavity and start of perfusion	See troubleshooting for step 26
		A non-fixative (e.g. PB) was used before perfusate	DO NOT use any non-fixative such as saline to “clear” the blood-vessels; this increases the duration of hypoxia of the brain and starts the apoptotic/necrotic process
47	Can only visualize and image dendrites that are less than 30 μm below the surface	Coverslip is slanted	Remount
		Objective spring is broken	Send in objective for repair
49	Dendrite appears to be moving across the screen during confocal imaging	Nail polish has not had enough time to harden or not enough nail polish has been applied	Because the recommended mounting media is aqueous and therefore never hardens, the stability of the slide prep relies on sufficient and hardened nail polish; wait 2–3 days before imaging; if movement is still seen, apply another coat of nail polish and wait another couple of days
49,58	Some dendrites or portions of dendrites appear fuzzy in the confocal image	Air bubbles in the immersion oil	Remove oil from slide and add a fresh amount of oil
		Air bubbles in mounting media	Remount
		Dendrite is under a blood vessel	Move on to the next segment; do not use for analysis
56–61	Problems with NeuronStudio		Consult the online manual http://research.mssm.edu/cnic/help/ns/index.html