Mouse anti neurofilament

Oocytes were fixed in 4% formaldehyde (Sigma-Aldrich, F8775) in ASW at 16 °C for 1 h under gentle stirring. After fixation, they were washed three times in PBS-Tw (0.1% Tween20 in PBS), permeabilized for 30 min in 0.1% Triton 100-X in PBS and washed three times in PBS-Tw. Oocytes were then incubated in blocking solution (0.1% Tween20, 0.5% BSA in PBS) for 30 min, followed by incubation in the primary antibody in PBS-BSA (1% BSA in PBS) for 24 h at room temperature and three washes in PBS-Tw. Incubation of the secondary antibody and phalloidin (1/200, Alexa FluorTM 488 Phalloidin, Invitrogen, A12379) was carried out for 24 h at room temperature. To label the maternal and paternal DNA, Hoechst (Fisher Scientific, 62249) was added at a final concentration of 5 µg ml⁻¹ for the final 10 min of incubation. After incubation, oocytes were washed three times in PBS-Tw and one time in PBS and mounted for imaging. The primary antibodies were mouse anti-neurofilament (1/400, Sigma-Aldrich, N5264, for labelling the myoplasm), rabbit anti-phospho-S6 (1/400, Cell Signaling Technology, 2211, for labelling the endoplasmic reticulum) and rabbit anti-phospho-Myosin light chain (1/500, Cell Signaling Technology, 3674S, for labelling phospho-Myosin). The secondary antibodies were goat anti-mouse/rabbit conjugated to Alexa Fluor 488/546/647 (1/200) Molecular Probes).

GnRH immunocytochemistry was performed in 1–2 year old male zebrafish that underwent a “mating-training”. This consisted in crossing the animals two times a week for two weeks. Fertile males were selected and sacrificed at 10 AM after being crossed. The brains were fixed in Bouin's solution for 24 h, embedded in paraffin and thin sections (7 µm) were obtained (Cortés-Campos et al., 2011 (link)). Alternatively, brains were fixed in PFA 4% with 7% saturated Picric Acid for 4 h at room temperature and thick cryostat sections (20 µm) were obtained (Harden et al., 2012 (link); Whitlock et al., 2003 (link)). Immunocytochemistry was carried out as previously described (Cortés-Campos et al., 2011 (link)). The primary antibodies used were: mouse anti-GnRH LHR13 (1:100; Park and Wakabayashi, 1986 (link)), mouse anti-GnRH HU11B (1:100; Millipore, Temecula, CA, USA; MAB5456), rabbit anti-GnRH BB8 (1:1000; Kah et al., 1986 (link)), rabbit anti-mGnRH (1:500; Sigma-Aldrich, St. Louis, MO, USA; G8294) and mouse anti-neurofilament (1:200; Sigma-Aldrich; N2787). Immunoreactivity was visualized using VECTASTAIN^® ABC Kit (Vector Laboratories Inc, Burlingame, CA, USA) for thin sections or Alexa-labeled secondary antibodies (1:500; Invitrogen, Rockville, MD, USA) for thick sections. The samples were DNA counter-stained with ferric hematoxilin or DAPI (1:1000; Invitrogen).