Anti gfp a6455

For experiments without EdU, ovaries were dissected directly into a fixation solution of 4% paraformaldehyde in PBS for 10 min at room temperature, rinsed 3x in PBS, and blocked in 10% normal goat serum (NGS) (Jackson ImmunoResearch Laboratories) in PBS with 0.1% Triton and 0.05% Tween-20 (PBST) for 1 h. Monoclonal antibodies for Fas3 were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. 7G10 anti-Fasciclin III was deposited to the DSHB by Goodman, C. and was used at 1:250 in PBST. Other primary antibodies used were anti-GFP (A6455, Molecular Probes) at 1:1000 in PBST. Ovaries were incubated in primary antibodies overnight, rinsed three times in PBST, and incubated 1–2 h in secondary antibodies Alexa-488 and Alexa-647 (ThermoFisher) at 1:1000 in PBST to label GFP and Fas3, respectively. DAPI-Fluoromount-G (Southern Biotech) was used to mount ovaries.

PMA and ionomycin were purchased from Sigma-Aldrich. TNFα was from CellGenix (Freiburg, Germany). PE-conjugated anti-mouse H2K^k (CL9005PE) was from Cedarlane (Ontario, Canada). Anti-GFP (A6455) was from Molecular Probes (Eugene, USA). Mouse anti-human CD3 (555336), -CD28 (555725), and -PKCθ (610089) were from BD Biosciences (San Jose, USA). Anti-CKIP-1 (D-20, sc-50225), -IKKα/β (H470, sc-7607), and -Lck (3A5, sc-433) were from Santa Cruz Biotechnology (Santa Cruz, USA). Anti-β-actin (AC-15, A5441), -c-Myc (9E10, M5546, and C3956), and -FLAG (M2, F3165) were from Sigma-Aldrich. Anti-HA (12CA5) was from Roche (Mannheim, Germany). Anti-p-Erk (Thr202/Tyr204, E10, #9106), -Erk (#9102), and -CARMA1 (1D12, #4435) were from Cell Signaling Technology (Danvers, USA).

For experiments without EdU, ovaries were dissected directly into a fixation solution of 4% paraformaldehyde in PBS for 10 min at room temperature, rinsed 3x in PBS, and blocked in 10% normal goat serum (NGS) (Jackson ImmunoResearch Laboratories) in PBS with 0.1% Triton and 0.05% Tween-20 (PBST) for 1 hr. Monoclonal antibodies for Fas3 were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. 7G10 anti-Fasciclin III was deposited to the DSHB by Goodman, C. and was used at 1:250 in PBST. Other primary antibodies used were anti-GFP (A6455, Molecular Probes) at 1:1000 in PBST. Ovaries were incubated in primary antibodies overnight, rinsed three times in PBST, and incubated 1–2 hr in secondary antibodies Alexa-488 and Alexa-647 (ThermoFisher) at 1:1000 in PBST to label GFP and Fas3, respectively. DAPI-Fluoromount-G (Southern Biotech) was used to mount ovaries.