CSHL Press, Cold Spring Harbor, NY, USA, 2003. Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5170 www.cshprotocols.org. Protocol.
Downloaded from http://cshprotocols.cshlp.org/ at UQ Library on February 2, 2016 - Published by Cold Spring Harbor Laboratory Press
Protocol
Alcian Blue/Alizarin Red Staining of Cartilage and Bone in Mouse Dmitry Ovchinnikov INTRODUCTION The vertebrate skeleton forms by endochondral and intramembranous bone formation. During endochondral bone formation, mesenchyme condensations give rise to cartilages that are eventually replaced by bone. However, there are some permanent cartilages that do not ossify, such as the cartilage of the trachea and articular cartilage of the joints, and intramembranous bone formation occurs directly without a cartilage template. This article describes a method for simultaneously visualizing cartilage and mineralized tissues (notably bone) in the developing mouse. It makes use of alcian blue and alizarin red stains, and it is best used for later fetal, newborn, and early post-natal stages of development. Neonatal skeletons are especially well suited for this technique.
RELATED INFORMATION A method to reveal the cartilaginous skeleton of embryos between 12.5 and 16.5 days post-coitum (dpc) is described in Alcian Blue Staining of the Mouse Fetal Cartilaginous Skeleton (Nagy et al. 2009a). For staining the bone of newborn and adult mice, see Alizarin Red Staining of Post-natal Bone in Mouse (Nagy et al. 2009b).
MATERIALS CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with , and recipes for reagents marked with .
Reagents Acetone Alcian blue stain (m) Alizarin red stain solution, freshly prepared Ethanol (70% and 95%) Glycerol:ethanol (1:1) Mice (fetuses or neonates) Potassium hydroxide, 1% (w/v) in H2O 1% potassium hydroxide/20% glycerol Tap water, 65ºC-70ºC
Equipment Dissecting microscope and photographic equipment for documentation Euthanasia equipment Forceps and scissors (size depends on age of fetus) Tubes (50-mL screw-cap, clear plastic) or tissue culture dish (six-well) (see Step 6)
Adapted from Manipulating the Mouse Embryo, 3rd edition (eds. Nagy et al.). CSHL Press, Cold Spring Harbor, NY, USA, 2003. Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5170 © 2009 Cold Spring Harbor Laboratory Press
www.cshprotocols.org
1
Vol. 4, Issue 3, March 2009
Downloaded from http://cshprotocols.cshlp.org/ at UQ Library on February 2, 2016 - Published by Cold Spring Harbor Laboratory Press
METHOD 1. Isolate the fetuses or euthanize neonates according to local regulations. Isolated fetuses or euthanized neonates can be stored in tap water at 4°C for 1-24 h if necessary. 2. Scald the fetuses or pups in hot tap water (65°C-70°C) for 20-30 sec. Carefully peel off the skin
with forceps. Scalding makes maceration of the tissue easier. 3. Use forceps to eviscerate the bodies, including the contents of the peritoneal and pleural cavities. 4. Fix the mice in 95% ethanol overnight. Be sure to remove all of the bubbles from the body cavity. 5. Transfer the mice to acetone and incubate overnight at room temperature to remove fat. 6. Rinse the specimens briefly in deionized H2O. Stain for cartilage by covering the bodies completely
with alcian blue stain (m). Leave the mice in the stain for 24 h.
Use a 50-mL screw-capped plastic tube, or for newborn mice or younger fetuses, use a six-well tissue culture plate. Be sure to remove all of the bubbles from the body cavity. 7. Wash the mice in 70% ethanol for 6-8 h. Include several ethanol changes. 8. Transfer the mice to 1% potassium hydroxide. Keep them in potassium hydroxide overnight or
until the tissues are visibly cleared. 9. Counterstain the bone by soaking the samples in alizarin red stain solution overnight. Younger fetuses need less time in the stain. 10. Clear the samples by placing them in 1% potassium hydroxide/20% glycerol for 2 d or more.
When the samples are appropriately cleared, place them in glycerol:ethanol (1:1) for documentation and storage (see Discussion). An example of a newborn skeleton stained with alcian blue and alizarin red is shown in Figure 1.
DISCUSSION Photodocumentation of skeleton preparations is best with bright-field optics and transillumination. The limbs can be removed from the skeleton preparation very easily with forceps. This facilitates visualization of the rest of the body or documentation of the limbs in isolation. The rib cage can be dissected away from the skeleton preparation by cutting the ribs close to the vertebrae. Place a glass slide on top of the isolated rib cage to create a flat mount for photodocumentation.
FIGURE 1. Alizarin red/alcian blue staining of a newborn skeleton. Ossified tissue (predominantly bone) stains red and cartilage stains blue. The soft tissues are cleared in an alkaline solution and glycerol. (Image courtesy of Kazuhisa Nakashima, University of Texas M.D. Anderson Cancer Center. For color figure, see doi: 10.1101/pdb.prot5170 online at www.cshprotocols.org.)
REFERENCES Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2009a. Alcian blue staining of the mouse fetal cartilaginous skeleton. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5169.
www.cshprotocols.org
Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2009b. Alizarin red staining of post-natal bone in mouse. Cold Spring Harb. Protoc. (this issue). doi: 10.1101/pdb.prot5171.
2
Cold Spring Harbor Protocols
Downloaded from http://cshprotocols.cshlp.org/ at UQ Library on February 2, 2016 - Published by Cold Spring Harbor Laboratory Press
Alcian Blue/Alizarin Red Staining of Cartilage and Bone in Mouse Dmitry Ovchinnikov Cold Spring Harb Protoc; doi: 10.1101/pdb.prot5170 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Cell Biology, general (1158 articles) Developmental Biology (594 articles) Laboratory Organisms, general (885 articles) Mouse (355 articles) Phenotypic Analysis (35 articles) Visualization (475 articles) Visualization, general (345 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
