Ab14206

Whole-mount immunofluorescent staining was performed according to the previously published protocol [32 (link)]. Prior to immunofluorescent staining, gonadal fragments were permeabilized in a 0.5% solution of Triton X100 in 1× PBS for 4–5 h at RT followed by washing in 1× PBS at RT. The following primary antibodies were used: rabbit polyclonal antibodies DDX4 antibody (C1C3, GeneTex Inc., Irvine, CA, USA) against Vasa protein; rabbit polyclonal antibodies against SYCP3 (ab14206, Abcam, Cambridge, UK); chicken polyclonal antibodies against SYCP1 protein (gift from Prof. Sean Burgess; [48 (link)]). After incubation for 1–2 h in a 1% blocking solution (Roche, Mannheim, Germany) dissolved in 1× PBS, primary antibodies were added for 12 h at RT. Secondary anti-rabbit antibodies conjugated with Alexa-488 fluorochrome and anti-chicken antibodies conjugated with Alexa 594 were applied for 12 h at RT. Washings from primary and secondary antibodies were carried out in 1× PBS with 0.01% Tween (ICN Biomedical Inc., Solon, USA). Tissues were stained with DAPI (1 mg/mL) (Sigma Aldrich, Saint Louis, USA) in 1× PBS at RT overnight.

SC spreads were prepared via drying-down technique described by Peters et al.^{34 (link)}. Immunofluorescence staining was performed according to the protocol by Anderson et al.^{35 (link)}. Prior to immunostaining, slides were incubated in a solution of 10% PBT (PBS with 3% bovine serum albumin and 0.05% Tween 20) and 90% PBS for 45 min to reduce non-specific antibody binding. Primary antibodies included rabbit polyclonal anti-SYCP3 antibodies (1:500; Abcam, ab15093), human anticentromere antibodies (1:100; Antibodies Inc., 15-234) and mouse monoclonal anti-MLH1 antibodies (1:30, Abcam, ab14206). The slides were incubated with antibodies overnight in a humid box at 37 ℃, and then washed three times in PBS with 0.1% Tween 20 for 15 min each time. Secondary antibodies included Cy3-conjugated goat anti-rabbit (1:500; Jackson ImmunoResearch, 111-165-144), FITC-conjugated donkey anti-human (1:100; Jackson ImmunoResearch, 709-095-149) and FITC-conjugated goat anti-mouse (1:30; Jackson ImmunoResearch, 115-095-003). The slides were incubated with them for 1 h under the same conditions. After washing, the slides were mounted in Vectashield medium with DAPI (Vector Laboratories, cat No. H-1000-10) under the coverslips. Microscopic analysis and image processing were performed as described previously^{36 (link)}.