Ddx4 antibody c1c3

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.

Kinetochore proteins were detected by human CREST serum (Antibodies Incorporated), and Vasa was detected with the rabbit polyclonal DDX4 antibody [C1C3] (GeneTex) via 3D immunofluorescence staining. Prior to the addition of the antibodies, the tissues were incubated in a 0.5% solution of Triton X100 in 1× PBS for 4–5 hours at RT, washed in 1× PBS at RT and incubated for 1–2 hours in a 1% blocking solution (Roche) in 1× PBS. Incubation with the primary antibodies (concentration 1:40) was carried out at RT overnight, followed by washing in 1× PBS with 0.01% Tween (ICN Biomedical Inc). Secondary antibodies conjugated with the Alexa-488 fluorochrome were applied for 12 hours at RT. The tissues were then washed in 1× PBS with 0.01% Tween (ICN Biomedical Inc) and stained with DAPI (1 µg/µl) (Sigma) in 1× PBS at RT overnight.

Gonads of fish larvae were isolated and fixed in 2% PFA in 1×PBS followed by washing in 1×PBS. Tissues were stored until usage in 1×PBS with the addition of 0.02% sodium azide. Prior to immunofluorescent staining, gonadal fragments were permeabilized in a 0.5% solution of Triton X100 in 1×PBS for 4–5 hr at RT and washed in 1×PBS at RT. The following primary antibodies were used: rabbit polyclonal antibodies DDX4 antibody (C1C3, GeneTex) against vasa protein; rabbit polyclonal (ab14206, Abcam) against SYCP3 protein and chicken polyclonal against SYCP1 protein (a gift from Sean M. Burgess). After incubation for 1–2 hr in a 1% blocking solution (Roche) dissolved in 1×PBS, primary antibodies were added for 12 hr at RT. After three times washing in n 1×PBS with 0.01% Tween (ICN Biomedical Inc), secondary antibodies Alexa-488-onjugated goat anti‐rabbit IgG (H+L) (Invitrogen) and Alexa‐594‐conjugated goat anti‐chicken IgG (H+L) (Invitrogen) were added for 12 hr at RT. After three times washing in 1×PBS with 0.01% Tween (ICN Biomedical Inc) tissues were stained with DAPI (1 mg/ml) (Sigma) in 1×PBS at RT overnight.