Protocol for the Primary Culture of Cortical and Hippocampal neurons
Solutions and media required:
Poly D-lysine/laminin solution - pdf
DM/KY - pdf
Optimem - pdf
Neuronal growth medium - pdf
Set-up for the dissection:
Day 1:
Add poly D-lysine/laminin solution to culture dishes/coverslips. Swirl the plate to ensure that the coating mix covers the entire bottom of the plate. Leave the dishes/coverslips in the 37oC/5% CO2 incubator overnight.
Day 2:
Wash the dishes/coverslips twice with sterile water; remove the final wash and leave them liquid-free in the incubator.
Make up DM/KY, sterile filter and place on ice
Make up the trypsin inhibitor solution and the papain solution BUT DO NOT add papain at this point; place solutions on ice.
Pour ice-cold DM/KY solution into several culture dishes: 1 large dish for the pups and 10cm dishes for the pup heads, for the intact brains and for the dissected cortices. Place dishes on ice.
Obtain pregnant rat
Dissection of hippocampus and cortex:
Sacrifice the rat by placing a plastic dish containing dry ice in the cage and then pouring water into this plastic dish; cover the top of the cage with a bag.
After the rat fails to move spontaneously or in response to pain (touch the eye and look for a reflex), incise along the abodmen and remove the uterus. Place the pups into the large culture dish.
Remove the heads of the pups and place in a 10cm dish
For each head, remove the skin and cut along the scalp in the midline with fine scissors. Make a similar midline cut in the calvarium. Deflect the calvarium with a blunt spatula and scoop the brain into another 10cm dish containing ice-cold DM/KY.
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