小鼠实验取材要点

Tissue Harvest Protocol

Set-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 pads

sac pack including: 1 pairs of hemostats

1 scissor

1 forcep

2-4 microclamps (s ize 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 lyse Put blood into eppendorf with 5ul of heparin 1000Units/ml. (if collecting plasma no heparin for serum)

*see below steps for individual blood collection circumstances

After cardiac puncture remove other organs as gently and promptly as possible.

For Serum:

No Heparin during collection at all

Use 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 cells Let sit in 4︒ overnight (for clotting).

Next day spin blood @ 3000 RPM @ 4︒ for 10 min

Apportion 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 sucking up of blood

1 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 initials

For Plasma:

Heparin is used @ 1000 USP Units/ml

*Check Experiment form for type of heparin needed to ensure good test results Spin plasma down @ 3000 RPM @ 4º 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 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 initials

*NOTE: When using the Heska machine for analysis, DO NOT Collect plasma w/ Na Heparin or EDTA tubes if analyzing electrolytes such as: K, Mg+,Na+

Use Li Heparin tubes instead

Organ 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 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 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.