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Mouse tissue collection and assessments

DS Dominik Saul
JF Joshua N Farr
SK Sundeep Khosla

Last updated date: Sep 6, 2022 Views: 539 Forks: 0

original An abbreviated version of this protocol was published in eLife in Oct, 2021
Modulation of fracture healing by the transient accumulation of senescent cells
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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)

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Saul, D, Farr, J and Khosla, S(2022). Mouse tissue collection and assessments. Bio-protocol Preprint. bio-protocol.org/prep1904.
  2. Saul, D., Monroe, D. G., Rowsey, J. L., Kosinsky, R. L., Vos, S. J., Doolittle, M. L., Farr, J. N. and Khosla, S.(2021). Modulation of fracture healing by the transient accumulation of senescent cells. eLife. DOI: 10.7554/eLife.69958

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