Fractionation of synaptosomes

Pre-chill the homogeniser and buffers on ice. Weigh the tissue

Add 4x volume of ice-cold homogenizing buffer to glass homogenizers

Apply 12 strokes of even tension with the homogeniser rod

Transfer to 1.5ml tube (original tube)

Determine which tissue has the smallest volume and use that volume for all samples

Centrifuge at 1,000g for 10mins

Transfer the supernatant into fresh 1.5ml tube and spin at 12,500g for 15mins

Discard supernatant carefully

Resuspend pellet in 1ml of H-buffer, transfer to fresh glass homogenizer and mash for 6 strokes even tension

In polypropylene/ultra-clear tube add 5ml of 1.2M sucrose

Slowly and steadily add in 5ml of 0.8M sucrose to create clear/distinct gradient

Overlay sample on top of gradient

Weigh sucrose gradient tubes (add H-buffer if weight is off by more than 0.05g)

Centrifuge with slow accleration and zero brake in 4 degrees at 23,600g for 70 mins in a SW41 rotor

Using syringe, extract synaptosome layer between 0.8M and 1.2M sucrose and transfer into fresh 1.5ml tubes

Plate sample with appropriate dilutions into 24-well plate (dilute with H-buffer or PBS)

Spin in 4°C, max speed ~4,000 rpm for 20mins

Remove liquid and fix in 4% PFA for 20mins on rocker/shaker for 20mins

Wash x2~3 with PNBS

Either cover and place in fridge or move directly to immunofluorescence protocol