Goat anti mouse alexa 555

The antibodies used in this study included mouse anti-Flag (Sigma, 1:3000), rabbit anti-Flag (Sigma, 1:2000), rabbit anti-Myc (MBL, 1:3000), mouse anti-GFP (Abmart, 1:5000), mouse anti-GAPDH (Sungene Biotech, 1:5000), mouse anti-α-Tubulin (Sungene Biotech, 1:5000), rabbit-anti-Oct4 (Santa Cruz, 1:2000), rabbit anti-HA (MBL, 1:3000), HRP-conjugated goat anti-mouse (Sigma, 1:3000), HRP-conjugated goat anti-rabbit (Sigma, 1:3000), goat anti-mouse Alexa555 (Molecular Probes, 1:2000) and goat anti-mouse Alexa488 (Molecular Probes, 1:2000).

Ovaries were prepared for immunohistochemistry, as described previously [7 (link)]. The following primary antibodies were used: rabbit anti-GFP (1:500, Abcam, Cambridge, MA, USA); mouse anti-Hts (1:100, DSHB, Iowa, IA, USA); mouse anti-Orb (1:200, DSHB); rabbit anti-Vasa (1:500, Yeasen, Shanghai, China). The following secondary antibodies were used at a 1:1000 dilution: goat anti-rabbit Alexa 488; goat anti-mouse Alexa 555 (Molecular Probes, Abcam, Cambridge, MA, USA). DAPI dye (Yeasen, Shanghai, China) was used to visualize cellular nuclei. All samples were examined with a Leica fluorescent microscope. All micrographs were taken with an Olympus confocal FV1000 microscope (Tokyo, Japan), and Z-stacks were obtained for presentation.

Testes were prepared for immunostaining as previously described^{17 (link), 23 (link)}. Primary antibodies were diluted as follows: rabbit anti-GFP (1:500, Invitrogen); mouse monoclonal anti-Hts antibody (1:100, DSHB); rabbit anti-Vasa (1:500, Santa Cruz). Secondary antibodies goat anti-rabbit Alexa 488; goat anti-mouse Alexa 555 (Molecular Probes) were used at a 1:1000 dilution. FITC-conjugated Phalloidin (1:200, Beyotime) was used to visualize F-actin. All samples were examined using a Leica fluorescent microscope, and micrographs were taken using an Olympus confocal FV3000 microscope.

An average of 8000 cells sorted by FACS (GFP⁺/GFP⁻ and YFP⁺/YFP⁻) were independently plated on Poly-D-lysine hydrobromide (Sigma) coated Labteck, cultured for 90 min in Neurobasal medium with B27 supplement and 20% FBS. Cells that attached were fixed with 4% paraformaldehyde in PBS for 10 minutes, followed by successive washes with PBS and PBS-Tween (1X, 0.1% Tween). After incubation in blocking solution for 1 h (PBS-Tween 10% goat serum), cells were immunolabelled overnight at 4 °C using the following antibodies: mouse anti-TH (1:400; Millipore MAB318), rabbit anti-GFP-YFP (1:750; Megaprob A11122), rabbit anti-TPH2 (1:500; Novus Biologicals). The next day, samples were washed, then incubated during 1 h at room temperature with the following secondary antibodies: goat anti-rabbit Alexa 555 (1:1000; Invitrogen A-21428), goat anti-rabbit Alexa 488 (1:1000; Invitrogen A-11008), goat anti-mouse Alexa 555 (1:1000; Life Technologies A21425). Nuclei were labelled with Hoechst. All images were collected on a Leica microscope.

Third-instar Drosophila larvae were dissected in fresh HL-3 solution and fixed in 3.7% formaldehyde for 20 min at room temperature. After washing with PBS, tissue was permeabilized with PBS containing 0.4% TritonX-100 (PBT) for 1 h, followed by blocking in 1% BSA for 1 h at room temperature. Immunostaining was performed with the following primary antibodies: DAKO anti-total tau rabbit pAb (DAKO, A0024, 1:1,000), anti-CSP2 mouse mAb (DSHB, 6D6, 1:1,000) and anti-GFP rabbit pAb (Invitrogen, A-11122, 1:1,000) for probing LifeAct-GFP. The secondary antibodies used were: goat anti-rabbit Alexa 488 (Invitrogen, 1:5,000) and goat anti-mouse Alexa 555 (Invitrogen, 1:5,000). Following antibody labelling, the preparations were washed in PBT and mounted in Vectashield (Vector Laboratories). Samples were visualized on a Nikon confocal microscope or a Zeiss ELYRA super resolution microscope.

The cells were permeabilized with 0.2% Triton X-100 and then treated with primary antibodies: rabbit anti-EV1 [28 (link)] for EV1 or monoclonal mouse Enterovirus clone 5-D8/1 antibody (Dako) for CVB3 and CVB1. After 1 h of incubation, excess primary antibody was washed with PBS and cells were treated with secondary antibodies: goat anti-rabbit Alexa 555 (Invitrogen) or goat anti-mouse Alexa 555 (Invitrogen). Secondary antibody was washed with PBS and nuclei stained with DAPI in PBS. Finally, the cells were mounted into mowiol-DABCO.
Samples were imaged with Olympus FV1000-IX81 confocal microscope using 543-nm HeNe and 405-nm diode lasers. The imaging was carried out with 60× UPLSAPO objective (NA 1.35), and levels for the laser power and detector amplification were optimized using negative infection control.