Anti rat alexa568

Embryos where dechorionated in bleach and fixed in a 1:1 mixture of heptane and 40% formaldehyde for 9 min. Embryos were then stuck to tape, manually devetillinized in PBS and transferred to eppendorf tubes. Staining to quantify maternal KD and for SIM were carried out in the same way: embryos were blocked in 1% BSA for 1 h, incubated with primary antibodies overnight at 4C, washed in PBS-Triton-X 0.1%, incubated with secondary antibodies for 2 h at room temperature, washed in PBS-Triton-X and mounted in Vectashield mounting medium. Primary antibodies were: 1:100 rat anti-DCAT-1 [Developmental Studies Hybridoma Bank (DHSB)], 1:10 mouse anti-NECD (C458-2H, DSHB) and 1:10 rat anti-DCAD2 (DCAD2, DSHB). Secondary antibodies were: 1:200 anti-Rat-FITC (712-095-153, Jackson ImmunoResearch) for α-Cat KD quantification; 1:200 anti-Mouse-Alexa488 (AB_2338189, Invitrogen) and 1:200 anti-Rat-Alexa568 (A-11077, ThermoFisher) for SIM. Embryos were also stained with 1:1500 Phalloidin-iFluor647 (ab176759, Abcam).

Indirect immunofluorescence was performed as described [35 (link)]. The primary antibodies are anti-mCherry monoclonal antibody (16D7) at 1:200 dilution (Thermo Fisher, MA) and anti-α-tubulin antibodies (clone B-5-1-2, Sigma-Aldrich) at 1:5000. The secondary antibody was anti-rat-Alexa 568 (Thermo Fisher, MA) at 1:1000 and DyLight 405 goat anti-mouse antibody (BioLegend, CA) at 1:2000, respectively. Cells were stained with 1 μM or 0.5 μM Sytox Green (Invitrogen, CA). Images were collected using an inverted Zeiss LSM 510 Laser Scanning confocal microscope. Images in S1 Fig were collected using DeltaVision microscope. Images were exported in TIFF format, merged and analyzed using ImageJ (NIH).

Mouse or goat IgG directed against TLR9 or Cav-1 and rabbit IgG directed against TLR9, Cav-1, GAPDH, TLR4, IRAK-4, TRAF6, TRAF3, MyD88, IRF7, IRF3, p38MAPK and JNK were obtained from Abcam (Abcam, USA). Anti-Ly6G and anti-CD11b were from Abcam Plc (Cambridge, UK). Mouse TNF-α (RAB0477) and IL-6 (RAB0308) ELISA Kits were purchased from Sigma (Sigma-Aldrich China, Shanghai). MyD88 homodimerization inhibitor peptide (NBP2-29328) was purchased from Novus Biologicals (Novus, USA).
MPLA (L 5399) was from Sigma (Sigma, USA) TLR9-specific shRNA (sc-40270-SH), Cav-1 shRNA (sc-29241-SH) and transfection reagent (sc-108061) were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-TLR9 for flow cytometry (560425, BD), Anti-Cav-1 for Flow Cytometry (PA1-064, Invitrogen), anti-MyD88 for flow cytometry (566354, BD), anti-IRF3 for flow cytometry (566347, BD), anti-TRAF3 for flow cytometry (PA5-29091, Invitrogen) and Fugene HD transfection reagents were obtained from Promega (Promega, USA). The full-length Cav-1 was ligated into pECFP-C1 and TLR9 was inserted into pEYFP-N1. Anti-mouse-IgG3-FITC was from Southern Biotech (Birmingham, USA); anti-human IgG-FITC, anti-rat Alexa568, and anti-rabbit Alexa555 were purchased from Thermo Fisher Scientific (Braunschweig, Germany); anti-FITC-Alexa488 was from Life Technologies (Darmstadt, Germany); and DAPI was from Sigma (Deisenhofen, Germany).