Vectashield medium h 1200 with dapi

Fixation and F-actin staining of Drosophila testes were performed as described in [14 (link)]. Fixation for the other immunostainings was performed as previously described [15 (link),16 (link)]. The primary antibodies and the dilutions (in PBS) used were as follows: Anti-tub (1:1000) (Sigma-Aldrich, St. Louis, MO, USA), anti-Pav (1:100) [17 (link),18 (link)], anti-Spd2 (1:5000) [19 (link)], anti-Feo (1:50) [20 (link)], anti-HTS (IBI) (1:5) (Hybridoma Bank, The University of Iowa, IA, USA) [21 (link)], anti-anillin (1:1000) [22 (link)]. The secondary antibody incubation was performed using both the FITC-conjugated anti-mouse IgG+IgM (1:20 in PBS; Jackson ImmunoResearch Laboratories, Cambridge, UK) and Alexa 555-conjugated anti-rabbit IgG (1:300 in PBS; Molecular Probes, Eugene, OR, USA) for 2 h at room temperature. Slides were then mounted in Vectashield medium H-1200 with DAPI (Vector Laboratories, Burlingame, CA, USA) to stain DNA and reduce fluorescence fading.
Slides with mitotic chromosome preparations and fixed testes were analyzed using a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Obezkochen, Germany), equipped with a cooled CCD camera (Photometrics, Woburn, MA, USA). Gray-scale digital images were collected separately, converted to Photoshop format, pseudocolored, and merged.

Fixation and staining were performed on dissected third-instar larvae testes as previously described [8 (link)]. The primary antibodies and the dilutions used were as follows: Anti-tub (1:1000) (Sigma-Aldrich, St. Louis, MO, USA), anti-DmATPCL (1:100) [14 (link)], anti-Lava lamp (1:300) [16 (link)]. The secondary antibody incubation was performed using FITC-conjugated anti-mouse IgG + IgM (1:20; Jackson ImmunoResearch Laboratories, Cambridge, UK), Alexa 555-conjugated anti-rabbit IgG (1:300 in PBS; Molecular Probes, Eugene, OR, USA) and Alexa Fluor 488-conjugated anti-guinea pig IgG (1:300 in PBS; Jackson laboratories) for 2 h at room temperature. Slides were then mounted in Vectashield medium H-1200 with DAPI (Vector Laboratories, Burlingame, CA, USA) to stain DNA and reduce fluorescence fading. Slides were analyzed using a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Obezkochen, Germany), equipped with a cooled CCD camera (Photometrics, Woburn, MA, USA). Gray-scale digital images were collected separately, converted to Photoshop format, pseudo-colored, and merged. Quantification of Lva spot was carried out using the ImageJ software. Box plots were obtained with Python 3.3.