Back to Manual - Perfusion
Mix and store all four solutions ahead of time according to the worksheets. Store the worksheets with the containers of solution in refrigerator. Shelf life is about one year. They only need to be refrigerated so that the solution will be cold when needed, but not to maintain quality.
Water, PBS, sodium azide 0.1%, heparin.
This is an isotonic buffered solution for washing out the blood. The sodium azide immediately halts mitochondrial respiration and the associated cascade of damage that would otherwise follow.
Perfuse at 100 mmHg. Only part of the volume will be used.
NBF 10%. Glutaraldehyde (GA) 3% is added at time of use.
Switch valves to the fixative container. Perfuse the entire 20 L.
Water, PBS, sodium azide 0.02%, Sodium Dodecyl Sulfate(SDS) 0.01%. GA 3% is added at time of use.
Formalin is not present in the carrier because it is not compatible with the DMSO in the CPA.
The SDS opens the BBB to allow CPA to move into the tissue without causing dehydration and mechanical distortion.
Switch valves to the carrier container. Start ramping in the CPA with the second pump.
Water, PBS, DMSO 37% v/v, Ethylene Glycol 37% v/v. Total 74% v/v, or 82% w/v.
GA 3% is added at time of use.
Set the software to follow this straight ramp. By the end of the ramp, it will be pumping 100% from the CPA container.
|Time||%CPA v/v||%CPA w/v||Brix||RI||%Target|