Alexa fluor 350 conjugated anti mouse secondary antibody

Slides with spermatocytes were washed in blocking solution (1% BSA, 0.05% Tween-20 in PBS) for 30 minutes and then incubated with primary antibody overnight in a humidified chamber at 4°C. Slides were washed multiple times in blocking solution and incubated with complementary Alexa Fluor conjugated secondary antibody for 2 hours at RT. Slides were extensively washed with blocking solution and in the last washing step DAPI was used as a counter stain (unless stated otherwise). Slides were then mounted using Prolong Gold antifade reagent (Life Technologies). For double antigen staining, two primary antibodies of different origin were combined together in an overnight incubation. For triple antigen staining, two primary antibodies of different origin were combined together in the first overnight incubation, then washed and incubated with complementary secondary antibodies. Third antigen staining (mouse SCP3 in squashes, mouse γH2Ax in spreads) was incubated overnight and then visualized with Alexa Fluor 350 conjugated anti-mouse secondary antibody (Life Technologies).