Mouse tissue collection and assessments

MMA Plastic embedding:

Week1:

• (Mon-Wed) cut unneeded metaphysis off; 1-3 days 100% ETOH changes 4C – we do this in cassettes to save time with solution changes
• (Thurs) Acetone 4C O/N
• (Fri) Xylene over weekend 4C, in a desiccator

Week2:

• (Mon) MMA1 stir 1hr, room temp. vacuum O/N; Make MMA2 stir 1hr, store 4C O/N (mix in the morning before use)
• (Tue) MMA2 room temp. vacuum 8hr; O/N 4C; Make MMA3 stir 1hr, store 4C O/N (mix in the morning before use)
•  (Wed) MMA3 1-2hr vacuum on ice; O/N 4C; Make MMA3 stir 1hr, store 4C O/N (mix in the morning before use)
• (Thurs) MMA3 2-4hr vacuum on ice; O/N 4C
• (Fri) Make embedding MMA3 Fresh, stir for 1hr at 4C, embed, N gas 5min, -20 for weekend
  • We superglue the bones in place to the bottom of the embedding molds to reduce movement.

Week3:

• (Mon) N gas 5min, 4C for 5 days
• (Fri or Following Mon) Remove from molds and air out in hood 3days

MMA solutions:

MMA1: 10mL/sample

• Methyl methacrylate          60mL – 360mL
• Butyl methacrylate             35mL – 210mL
• Methyl benzoate                5mL – 30mL
• PEG 400                              1.2mL – 7.2mL
  • Total                      about 100mL – 607.2mL

MMA2: 10mL/sample

• Methyl methacrylate          60mL – 360mL
• Butyl methacrylate             35mL – 210mL
• Methyl benzoate                 5mL – 30mL
• PEG 400                               1.2mL – 7.2mL
• Benzoyl peroxide                0.4g – 2.4g
  • Total                      about 100mL – 607.2mL

MMA3: 10mL/sample

• Methyl methacrylate          60mL – 360mL
• Butyl methacrylate             35mL – 210mL
• Methyl benzoate                 5mL – 30mL
• PEG 400                               1.2mL – 7.2mL
• Benzoyl peroxide                0.8g – 4.8g
  • Total                      about 100mL – 607.2mL

MMA3 for embedding: 3mL/sample

• Methyl methacrylate          60mL – 90mL
• Butyl methacrylate             35mL – 52.5mL
• Methyl benzoate                 5mL – 7.5mL
• PEG 400                               1.2mL – 1.8mL
• Benzoyl peroxide                0.8g – 1.2g
  • Total                      about 100mL –150mL
• Add 0.4mL (in 100mL) or 0.6mL (in 150mL) N,N dimethyl-p-toluidine just before embedding, enough time for a thorough mix is all that’s needed – 5 minutes or less

Catalog numbers:

• Methyl methacrylate (M55909-1L, Sigma)
• Butyl methacrylate (235865-1L, Sigma)
• Methyl benzoate (M29908-500g, Sigma)
• Polyethylene glycol 400 (PEG 400) (202398-500g, Sigma)
• Benzoyl peroxide (179981-500g, Sigma)
• N,N dimethyl-p-toluidine (41490-100mL, Sigma)
   

De-plasticizing:

3x 20min washes in 2-Methoxyethl Acetate (109886-1L, Sigma)