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小鼠实验取材要点.doc

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小鼠实验取材要点.doc
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Tissue Processing and Harvesting1Tissue Harvest ProtocolSet-Up:If plasma is desired use 1cc syringes w/ 25G needle coated w/ heparin (withdraw 1000U/ml heparin directly into syringe and pull plunger back and forth to coat inside of syringe. Expunge all the heparin from the syringe and the needle tip) REFER to EXPERIMENT FORM FOR EXPLICIT DIRECTIONS as to WHAT KIND of heparin is needed 1 metal tray large enough to fit mouse w/ board in blue padssac pack including: 1 pairs of hemostats1 scissor1 forcep2-4 microclamps (size doesn’t matter)cold PBS (use about 50ml/ animal)2% paraformaldehyde (use about 50ml/ animal)2-250ml lactated ringers solutions bags empty.Cut slit across one side at top of bag in order to fill with solution.Attach fitted tubing on end of bag (same tubing as hepatocyte tubing).Attach three way stopcock to end of tubing.Attach 18G needle to the stopcock.Fill one bag w/ cold PBS (depending upon how many mice you have to sac)Fill second bag w/2% para (depending upon how many mice you have to sac)Procedure:Hang bags on hooks provided in the hood. **Bag should be ~1meter (3ft) above surface level to get appropriate flow and pressure for perfusion.Shave abdominal area and remove excess hair with 4x4 gauze pad.Midline incision through skin and abdomen all the way up to the xyphoid(make sure to pick skin up when cutting to avoid organ damage)Cut diaphragm along rib cage to separate it Cut rib cage just lateral to the R & L internal thoracic arteries. Place hemostats on cut xyphoid segment with curvature of hemostat face up to hold for viewing heart for cardiac puncture.Perform cardiac puncture: Use 25G 5/8 needle with 1 ml syringe for cardiac puncture(mice)Use 18G needle w/ 5ml syringe for cardiac puncture (rats)Insert needle into R ventricle of heart and remove blood*Allow blood flow to control suction of syringe. Pulling too fast will cause cells to lysePut blood into eppendorf with 5ul of heparin 1000Units/ml. (if collecting plasma no heparin for serum)
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