Immunostaining of Drosophila Testes

Testes were dissected from pharate adult males in PBS and transferred to a drop of PBS containing 5%-glycerol on a microscope slide. The testes were then squashed with a coverslip, snap-frozen in liquid nitrogen and fixed in chilled methanol for 3 min. The samples were rehydrated in PBS for 5 min, washed in PBS containing 0.01% Triton X-100 (PBSTx) for 10 min and incubated overnight in a humid chamber at 4 °C with the following primary antibodies: rabbit anti-Asl^{22 (link)} (1:16,000; recognizes the N-terminus of the protein), chicken anti-D-Plp^{23 (link)} (1:1000), rabbit anti-Ana1^{13 (link)} (1:5000; recognizes the C-terminus of the protein) and guinea pig anti-Cep135 (1:500). The slides were washed three times in PBSTx for 10 min, incubated with appropriate secondary antibodies at 1:500 dilution: goat anti-chicken IgY Alexa Fluor 488 (Catalog # A-11039), goat anti-guinea pig IgG Alexa Fluor 488 (Catalog # A-11073), goat anti-rabbit IgG Alexa Fluor 568 (Catalog # A-11011), goat anti-chicken IgY Alexa Fluor 647 (Catalog # A32933) from Life Technologies, in a humid chamber for 1 h at 25 °C, followed by three 10 min washes in PBSTx before mounting in Vectashield containing DAPI.