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Leave it in during the day or overnight
The following day, sections were washed 6 times for 5 mins each in 0.01 M PBS, and incubated with the appropriate secondary antibodies conjugated to AlexaFluor 488 (donkey anti-rabbit IgG-conjugated Invitrogen, CA, 1:500 in PBST), or AlexaFluor 647 (goat anti-rabbit IgG-conjugated, Invitrogen, CA, 1:500 in PBST) for 2 hours at room temperature in dark
In this way, you can be sure that all systems in your network really are recorded
