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.
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