P. Fajer page 1 of 2

Glycerinated Rabbit Psoas Fibers

Equipment:

Scalpels, forceps, plastic garbage bags (for asphyxiation of rabbit and disposal of remains), scissors, acrylic sticks 2-5mm thick and 20cm long, 100ml graduated cylinders (for storing tied fibers), string or thread, and squirt bottle of RB(KAc).

Method:

1. Prepare solutions, label and number 100ml graduated cylinders, get the above equipment ready for use.

2. Kill rabbit (CO2 asphyxiate for ~5 minutes), bleed, and gut. Bleed as fast as you can.

3. In cold room with carcass on ice use a fine forceps to strip outer fascia (membrane) from psoas. Separate the fibers gently into 2mm bundles.

4. Tie one end of the bundle to wooden stick. Cut bundle and allow to slacken before typing second end to the stick. Rinse with RB(KAc).

5. Put tied bundles into cold Fiber Glycerination Buffer (FGB). Place on shaker for 12-24 hrs (shake slow enough so the cylinders will not fall over).

6. Change bundles to Fiber Storage Buffer (FSB) and store for 24-36 hrs, changing the buffer at least twice per 24 hrs. Final storage is in the freezer (-20°C), the buffer should be changed immediately prior to placing the fibers into the freezer. Fibers stored at -20°C should be good for 6 months, but attempt to use within 3 months.

Solutions:

RB(KAc):

Final Concentration Addition per Liter

KAc 60 mM 24 ml of 2.5 M

MOPS 25 mM 25 ml of 1.0 M

MgCl2 2 mM 6 ml of 1.0 M

EGTA 1 mM 2 ml of 0.5 M

NaN3 1 mM 1 ml of 1.0 M

pH 7.0 adjust pH to 7.0 after mixing

FGB: 75% RB(KAc), glycerol, 0.5% Triton, + 9 mM EGTA (this is in addition to the 1 mM that is already present).

All salts are per aqueous volume!

FSB: 50% RB(KAc), 50% glycerol; for final storage add 0.1 mM DTT.

All salts are per aqueous volume!

Fiber Prep Data Summary

A new section (with tab) should be started for each rabbit in the fiber prep book. A new form (this sheet) for any fiber prep whether it is unlabeled or labeled should be filled out. Whenever you assay anything, put it in the prep notebook!

PARAMETER / DATE / RESULT (UL) / RESULT (L) / L/UL / COMMENTS
High Salt
ATPase / -Ca +Ca / IU
IU / IU
IU
-Ca +Ca
-Ca +Ca
Physiological
ATPase / -Ca +Ca
-Ca +Ca
-Ca +Ca
Tension / -Ca +Ca / kg/cm2
kg/cm2 / kg/cm2
kg/cm2
-Ca +Ca
-Ca +Ca
Sarcomere
Length
[Probes]/[Head]
f(SH1)
------
[Probes]/[Head]

Fiber preperation.doc 10/20/1999 4:55 PM10/20/1999 4:55 PM