Kingsley Lab protocol for Alcian blue + alizarin red skeletal preps (adult)

Ryan Rountree 3/31/03

Version 1.1

1) Make strings and labels for each of your skeletal preps

String is in the stock room with the office supplies. Cut into approx. 16 inch pieces. Make a slip knot on one end (for attaching to the animal) and fold a strip of tape across the other end (for your label)

2) Do any assays necessary prior to skeletal prep. Examples:

Weigh

Joint range of motion

Cut tail for genotyping or LacZ staining

Etc..

3) Skin & Eviscerate animals (you might save organs for histology or LacZ staining, but usually don't)

Have someone show you how to do this - there are quick ways!!

If you want to get nice staining everywhere, remove all excess fat. The most important place is behind the neck and between the clavicles

If you want the Alcian blue staining to penetrate into the finger/toe joints, you must do a good job of removing all the skin around them. Easiest way is to start the skinning process but leave the skin on the hands and feet (as usual). Then make a cut all the way along the dorsal skin of the hand/foot and then each finger. Then when you remove the skin from the animal, use forceps to get everything off the fingers (will come off in several pieces). Pinching right between the fingers and pulling the skin off helps a lot.

4) Use the slip knot to firmly tie string + label to the tail of the animal.

5) Put into beaker filled with 95% Ethanol for 1-2 days (cover beaker with tin foil throughout protocol)

I write on the tinfoil what I did on what day so I don't loose track

6) Replace ethanol with Alcian blue staining solution for 1-2 weeks (I standardized to 14 days)

MIX after the first day and every few days by sloshing the skeletons around

7) Pour Alcian blue off into the waste container under the hotroom sink. Add 95% ethanol for 1-2 days

8) Put skeletons into 1% KOH for several days (usually about 5?) until tissue is pretty clear

MIX after the first day and every 1-3 days after that. Replace with fresh 1%KOH is clearing is happening too slowly (happens if you have a lot of animals in too small a volume).

9) Put skeletons into fresh 1% KOH with alizarin red. and stain 1-3 days (or until staining complete)

MIX EVERY SINGLE DAY!!! If you don't staining will be totally uneven!

I have found that adding about 400ul 1% alizarin red for each skeleton in the beaker gives nice staining. But this is only approximate. Add stain every day until it's dark enough throughout bones.

10) Lift skeletons out of KOH and drain as well as you can. Put into new beaker with 100% glycerol. Cover with parafilm as best you can (prevents water in air from getting into glycerol). Leave overnight.

11) Pour off old glycerol and add fresh. Cover with parafilm. Leave overnight.

12) Put skeletons into labled 50 mL Falcon tubes with new glycerol (remove string).

Alizarin Red

Make 1% KOH in water

Add alizarin red powder to 1% final

Dissolve well (do NOT filter)

Alcian Blue staining solution

4 Volumes of 95% Ethanol

1 Volume of Acetic Acid

0.015% Alcian Blue 8GX (Sigma A-3157)

Stir up to 30 minutes - can take a while for it to all go into solution, and sometimes it just won't - don't worry about it and don't filter it! It will stain to faintly if you filter it.

NOTE: It is possible to do wholemount X-Gal staining of the skeleton prior to the skeletal prep. See my x19.0 on 5/2/00 for more details. Summary: Skin completely the mice and put into ice-cold PBS. Fix the in about 10X volume of 4% paraformaldehyde at 4 degrees for 4 hours (I did 4 skeletons in a 500 mL beaker and filled with fix up to the 500mL mark). Rinse once quick with cold PBS, then 2X 30 min cold PBS. Rinse 1 hour in cold Xgal wash buffer. Stain 1-2 days at 4 degrees or room temperature (more chance for background stain at room temp, I did 4 deg for 1 day, room temp for 1 day) on rotating platform. (I used 275 mLs staining solution with 2.75mLs 50mg/mL Xgal to cover all four animals in a small histology dish). After staining rinse quick with PBS then rinse 1-2 hours PBS then put into 95% ethanol for 1-2 days. Skip the Alcian blue part of the protocol. Proceed with KOH and alizarin red staining. Try 2% KOH and replace with fresh if takes a long time to clear tissue (tissue is extra tough from fixation).