1、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
2、) 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 doesnt matter)cold PBS (use about 50ml/ animal)2% paraformaldehyde (use a
3、bout 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/ col
4、d 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 r
5、emove 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
6、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 b
7、lood*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) *see below steps for individual blood collection circumstances Tissue Processing and Harvesting2After c
8、ardiac puncture remove other organs as gently and promptly as possible.For Serum:No Heparin during collection at allUse a 25G needle w/ 1ml syringe for blood withdrawal in mice (18G w/ 5ml syringe for rats)*take off the needle before putting blood into eppendorf to prevent lysing cellsLet sit in 4 o
9、vernight (for clotting).Next day spin blood 3000 RPM 4 for 10 minApportion serum into separate eppendorfs using glass Pasteur pipette (120 (ul) microliters each) dispose of pipette in sharps container *protein globules will prevent easy aliquoting you may need to spin it down several times due to su
10、cking up of blood1 eppendorf is stored 4 for ALT/AST level analysis (only need 50ul) The rest of the eppendorfs are stored -20 Freezer in hallway label boxes with mouse serum, the group name and #s in the box, & primary resident initialsFor Plasma:Heparin is used 1000 USP Units/ml *Check Experiment
11、form for type of heparin needed to ensure good test resultsSpin plasma down 3000 RPM 4 for 5 min (Can be spun immediately)Apportion plasma into separate eppendorfs using a glass Pasteur pipette (120 (ul) microliters each) dispose of pipette into sharps container.1 eppendorf is stored 4 for ALT/AST l
12、evel analysis (only need 50ul) The rest of the eppendorfs are stored -20 Freezer in hallway label boxes with mouse serum, the group name and #s in the box, & primary resident initials*NOTE: When using the Heska machine for analysis, DO NOT Collect plasma w/ Na Heparin or EDTA tubes if analyzing elec
13、trolytes such as: K, Mg+,Na+Use Li Heparin tubes insteadOrgan extraction for protein and RNA analysis:*This step should be performed as rapidly as possible due to rapid tissue degradation All organs are put into liquid N2 immediately after harvest.Harvest liver first. Use microclamp and clamp off to
14、 R lateral lobes of liver. Cut the lobes out and separate into two eppendorfs if requested. Harvest lung next. Use microclamp and clamp off R lobes of lung lateral to the pulmonary branch. If you clamp too close to the main branch the L lobe tends to inflate during perfusion instead of being cleared
15、 of blood.Harvest spleen next. Cut away from fascia .Next harvest the kidney. Clamp the renal vessels with a microclamp and cut out the kidney.Harvest gut. Find Ileum. Move backwards about 2” from ileum and take a 1” section. Tissue Processing and Harvesting3Start Perfusion w/ PBS to remove all red
16、blood cells from remaining organs. Be sure to let the fluid run through the line to remove all the air before perfusing the animal.Insert 18G needle into L ventricle and turn stopcock to flow through the animal. Let liquid flow until the organs turn pale the fluid should escape through the cardiac p
17、uncture in the R ventricle.Next, start perfusion w/2% paraformaldehyde. Insert the 18G needle into the same spot as previous needle in L ventricle. Collect organs required and place in 10-20 mls of 2% paraformaldehyde in a 50ml conical.Samples for Immunoflurescences (IF), shall be incubuated in 2% p
18、araformaldehyde at 4C for 2hrs. Duration of fixation depends on tissue size. A lobe of mouse liver needs 2 hrs a lymph node requires an hour. If sample is left in fixitative too long, your sample will have higher autofluorescences and antigen access to your antibody of interest may be reduced. Sampl
19、es fixed in 2%paraformaldehyde may be processed for IF, Immunohistochemistry/H&E. The use of formalin is discouraged as it reduces future use of samples in IF experiments.After fixation, samples for IF are to be incubated at 4C in cold 30% sucrose for 24hrs. Changing the sucrose 3X within 24hrs furt
20、her minimizes paraformaldehyde negative effects.Samples are slow frozen by liquid nitrogen cooled 2-methylbutane (see detailed protocol at http:/www.cbi.pitt.edu/protocols/fixation.)Samples for H&E (see the Research Histology Service under the heading of Histopathology/Immunohistochemistry at http:/stiresearch.health.pitt.edu/content.) Tissues for H&E should be fixed in paraformaldehyde for a duration dependent of tissue size. Allow 1mm/hour for the fixative to penetrate your tissues and a volume of 10-20 times fixative volume to tissue. Once fixation complete, switch to 70%EtOH.
