User:Robert J. Schiemann/Notebook/Lipsick Lab/Protocols/Testes Dissection

From OpenWetWare
Jump to: navigation, search
  • Dissect testes in 1x PBS Solution. Store in eppendorf with 1x PBS until dissections are finished.
  • Fix with 4% Formaldehyde PBS Solution
    • 20-40 minutes (20 minutes optimal)
  • Wash 3 x 5 minutes in 1x PBS-T
  • Incubate 30 minutes with PBS 0.6% Triton.
  • Wash once with 1x PBS-T

If antibody staining, perform the following:

  • Block in 2% NGS
    • 1hr at RT or O/N at 4C
  • Incubate with primary antibody in 2% NGS
    • O/N at 4C or 2+ hours at RT
  • Wash 3 x 15 minutes in 1x PBST
  • Incubate with secondary antibody in 2% NGS (1:500)
    • 2hr at RT or O/N at 4C
  • Wash 3 x 15 minutes in 1x PBST

Final DNA staining:

  • Stain with ToPro3 1:1000 in 2% NGS
    • 30 mins at RT
  • Wash 4 x 5 minutes in PBST
  • Mount in Vectashield

Note: Whenever making a solution with NGS, centrifuge the NGS for a few minutes prior to use. This removes denatured proteins from the NGS which causes nonspecific binding of the antibody.