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


 * 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.