Brain Slice Preparation

Tips from one of the CALM’s previous directors DeLaine Larsen:

Cutting Slices:

•  300-400 um thick live slices for patching and then do the histology.
• 100um for in vivo tracer injections and for the reconstructions
• Note: The trick is the thicker slices are better for following axons in the tissue but you have to clear the tissue or the light scattering makes it impossible to image that deep

To clear Tissue

For sections mounted on slides

• Standard histology clearing
• EtOH series up to 100% with 2x in 100%
• Followed by Xylenes to defat.
• For some animals with more myelin than mice there would also be a chloroform step
• Note issue with Xylene is if there is anywater it will precipitate
• Then to Coverslip with DPX
• NO COVERSLIPPING with permount - this is ann old histology mountant it has autofluorescence
• Free floating
• Use the special nets with teflon sleeves and then a metal mesh, that stand up to xylenes
• Then I would go EtOH series to 100%, Xylenes
• Then ETOH series back down to rehydrate and then one of the glycerol-based mounting media