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