Skeletal analysis of mice
Most bones are formed from the cartilage primordium, which process is termed endochondral ossification. Therefore, impairment of cartilage development caused skeletal defects, including dwarfism. Cartilage contains a large amount of proteoglycans (PGs). Genetic mutations that affect biosynthesis of proteoglycan core proteins and glycosaminoglycans often impair cartilage development, causing skeletal defects. Examples are cartilage matrix deficiency, a natural knockout mouse of aggrecan core protein, perlecan-null mice and their human counterpart Schwartz-Jampel syndrome. In addition, knockout mice of PAPS synthase-2, brachymorphic, bm/bm, and nucleotide-sugar transporter SLC35D1, exhibit skeletal defects. As mutations of genes involving PGs and glycosaminoglycans may cause skeletal defects, analysis of skeleton is an initial step of the mutant mice. This section describes both gross and histological analysis of mice skeleton.
alcian blue 8GX (Sigma), preparation: 80 ml ethanol, 20 ml acetic acid, 15 mg alcian blue
alizarin red S (Chroma), 0.001% alizarin red S in 1% KOH
K-CX decalcifying reagent (Falma)
Anti-collagen II antibody, rabbit polyclonal (RDI)
Anti-aggrecan antibody (12/21/1-C-6, DSHB)
Anti-link protein antibody (9/30/8-A-4, DSHB)
Bovine testicular hyaluronidase (Sigma)
Chondroitinase ABC (Seikagaku Biobusiness)
HistomouseTM kit (Zymed, currently available at Invitrogen)
Antibody diluent with background reducing components (DAKO, S3022)
LSABTM2 biotinylated link for streptavidin HRP/AP (DAKO, K1015)
LSABTM2 streptavidin-HRP (DAKO, K1016)
Liquid DAB+ substrate chromogen system (DAKO, K3468)
10% goat serum (Nichirei)
Double staining with alcian blue and alizarin red for skeleton
Remove organs and skin as much as possible.
Fix mice in 96% ethanol.
Stain for 2-3 days in alcian blue solution.
Dehydrate for five days in 100% ethanol.
Immerse the specimen in 1% KOH for 3-5 days.
Stain the specimen in alizarin red solution for 2 days.
Destain alizarin red by immersing in 1% KOH for 1-2 days.
Replace to solution to water.
Transfer to 25% glycerin/water.
Gradually increase glycerin.
Store the double-stained specimen in 50-100% glycerin.
Immunohistochemical analysis of mouse skeleton
Fix the specimen with 10% buffered formalin.
Decalcify the specimen with K-CX overnight.
Embed the specimen in paraffin.
Prepare histology section slides at an appropriate thickness.
Deparaffinize the section slides.
Treat the section slides with 0.3~3% H2O2 in methanol for 10 min.
Wash with dH20.
Pretreat the section slides to retrieve antigen.
Wash with PBS for 5 min × three times.
Treat with 10% goat serum for 30 min~1 h at RT.
Wash briefly with PBS.
Treat with a primary antibody for 2 h at RT or 4 °C overnight in moist chamber.
Treat with biotinylated link and incubate for 20 min or longer at RT.
Treat with streptavidin HRP and incubate for 15 min or longer at RT.
Immunodetect with the Liquid DAB+ substrate chromogen system.