Table 2.

Timing by Experimental Task

Experimental Task		Hands On Time (in hours)	Variables Influencing Time	Overall Time in Culture
Coverslip Preparation	Application of Wax Spacers	~ 1 – 1.5 hours	Can be prepared ahead of time
	HCL Sterilization			Overnight
	Wash & Substrate Coating	1 hour over 3–3.5 hours (5 washes, 30 minutes each)
Neuronal Culture				13 Days
	Dissection & Cell Preparation	3.5 – 4.5 hours	Number of pups in the litter.
	Medium Changes	½ hour
Astrocyte Cultures				2 weeks +
	Primary Cell Dissection & Preparation	3 – 4 hours
	Medium Changes	½ to 1 hour every 2 – 3 days	Number of flasks
	Astrocyte passage for experiments	1.5 – 2 hours
	Serum Deprivation	½ hour to 1 hour	Number of wells	24 hours
	Treatment	~ 1 hour	Treatment preparation	24 hours *
Co-culture				24 hours *
Neuronal Fixation & Wash		¾ - 1hour
Immunolabeling	Day 1 – Primary AB	1.5 – 2 hours	Number of coverslips	Overnight (at least 18 hours)
	Day 2 – 2ndary Ab Washes and Incubation	2 ¾ - 3 hours
	Mounting Slides	1–1.5 hours	Number of coverslips
Imaging	Confocal Imaging	~ 3 – 4 hours per treatment group	15 neurons
	Thresholding	1 – 1.5 hours for 15 images
	3-D Image Analysis	1 – 1.5 hours per experiment