Phalloidin staining of whole mammary glands

Tuesday: 19.02.19.

For mice carrying the R26R-tdTomato allele:

Isolation

  1. dissect mammary glands from mice
  2. fix in 4% paraformaldehyde for 30-60min
  3. freeze tissue in OCT at -80°C or proceed directly to staining

Staining

  1. (wash away OCT with 37°C PBS, 2 x 15’)
  2. wash 4x 1hr with PBD0.2T
  3. wash 2x 1hr with PBS0.5T (PBS + 0,5% Triton-X100)
  4. Primary Abs, in fresh PBD0.2T (PBS + 1% BSA + 1% DMSO + 0.2% Triton-X100) o/n 4°C rocker
  5. wash 4x 1hr with PBD0.2
  6. Secondary Abs (1:333), DAPI (1:10.000)/TO-PRO-3 (1:1000), in fresh PBD0.2 o/n 4°C rocker
  7. wash 4x 1hr with PBD0.2T
  8. gentle rocker, 80% glycerol (PBS) for app. 1hr RT

Mounting

  1. for tdTomato allele: dissect under stereoscope with fluorescent lamp to reveal tomato-positive areas
  2. mount the samples between two converslips with 1-2 drops of 80% glycerol, use color-less silicon to seal the coverslips
  3. Samples can be stored at 4°C for some weeks or at -20°C for longer periods. Warm to RT before imaging to get best clearing effect.

Rios ACet al.Nature506p322-7(2014 Feb 20)

Primaries:

  • Basal marker, Keratin 14, PRB-155P (Rabbit -20°C) 1:500
  • Luminal marker, Keratin 8, DSHB Troma-I-C (mouse, 4°C) 1:500
  • Golgi marker, GM130, (mouse, -20°C) 1:500