MATERIALS

 
1. 4.5" x 5.5" x 1/4" cork boards
2. 1" metal straight pins
3. #2 lead pencil
4. 3 x 5 index cards
5. 1-2 gallon plastic bucket
6. Telly's or 10% formalin fixative
7. Brick or some other heavy weight
8. 70% EtOH in 500 ml wash bottle
9. Sharp nose scissors, small
10. Rat-tooth forceps
11. Plastic bag for disposal of carcass
12. Larger scissors
13. Anesthesia (pentobarbital)
14. 1 ml syringe and 23 or 25 gauge needle

METHOD

1. Kill mouse with anesthetic overdose or by cervical dislocation.
2. Pin mouse to cork board ventral side up by placing pins through base of tail and upper jaw.
3. Thoroughly wet the skin (fur) with 70% EtOH.
4. Make midline incision through the skin, using care not to penetrate the abdominal musculature, from external genitalia papillae at base of tail to top of the lower jaw.
5. Make parallel incisions in skin on both sides of external genitalia papillae from the top to the base of the tail.
6. Skin mouse by gently pulling the body wall away from the skin. This can be done by holding skin with rat-tooth forceps or finger while peeling body with other hand. The mammary fat pads will be exposed and remains with the skin.
7. Skin mouse in quadrants, starting with the inguinal region.
8. Pull skin flap as far from body as possible and pin to cork board; this maneuver will expose the #4 and #5 fat pads.
9. Gently dissect #5 fat pad away from the leg muscles. Pull leg through skin and cut off paw if necessary. Place pins through skin and cork board so that skin is taut.
10. Repeat maneuver on opposite side.
11. Free skin from thoracic region; pin skin to cork board to expose pads 1,2 and 3.
12. Pull limbs through skin; cut off leg at paws if necessary.
13. Pick up both hind limbs and sever spinal column at the tail base.
14. Carefully pull carcass away from skin by lifting and peeling towards head.
15. Snip the intrascapular brown fat away from the body to thoroughly free carcass from skin.
16. Snip skin from the head by cutting behind external ears, freeing the carcass from the skin.
17. Place pins through edge of skin to keep skin taut.
18. Discard carcass in plastic bag and dispose of appropriately.
19. Emerse skin and cork board in excess Telly's or 10% formalin fixative skin side down.
20. Use brick to totally immerse cork board in fixative. Fix at least overnight.
21. Discard fixative and wash in running water until most of the fixative is removed.
22. Use a No. 3 or 4 scalpel blade and lightly score the mammary fat pad side into quarters, making sure that the skin is not cut through; the right #1,2,3 fat pads should be contained in one quarter and the corresponding left side should be in another; the right #4 and 5 fat pads should be in one of the quarters and the corresponding left side in another quarter.
23. Using handle of scalpel holder, gently scrape mammary fat pads from skin.
24. Place the quarters into staining cassettes with the appropriate identification.
25. Place cassettes in acetone to defat at least overnight; one or two changes of acetone may be required.
26. process and stain whole mounts as follows, either manually or by autotechnicon.

1. 100% EtOH 1 hour
2. 95% EtOH 1 hour
3. 95% EtOH 1 hour
4. hematoxylin 1.5 hours
5. tap water wash 3-6 hours
6. 70% EtOH 1 hour
7. 95% EtOH 1 hour
8. 95% EtOH 1 hour
9. 100% EtOH 1 hour
10. 100% EtOH 1 hour
11. toluene 1 hour
12. toluene 1 hour

STOCK SOLUTIONS:

1. Stock Hematoxylin: 10 g in 100 ml 95% EtOH (let stand overnight before use)
2. Working hematoxylin solution: 1.6 g ferric chloride in 240 ml HOH 290 ml 1 N HCl 20 ml stock hematoxylin 1810 ml 95% EtOH
3. Instead of iron hematoxylin, Harris hematoxylin can be used.

27. Package and store in methyl salicylate; scintillation vials are ideal.