Goat anti rabbit igg alexa488 antibody

For each Western Blot, ~ 100 gemmules were grown in petri dishes with lake water containing 100 µg/mL ampicillin for 6–13 days at RT. Juveniles were scraped with a razor into 4× SDS-PAGE reducing loading buffer (1 M Tris, pH 7.0, 20% SDS, 20% Glycerol, 0.02% bromophenol blue and 2.5% β-mercaptoethanol), vortexed and boiled at 95 °C for 3 min. Proteins were separated by SDS-PAGE on a 10–12% gel, and transferred to a PVDF membrane (Millipore) at 350 mAmp for 30 min. Membranes were blocked for 1 h at RT in 5% nonfat milk in 1× PBST, pH 7.4 (0.05% Tween 20) and then incubated with affinity purified antibodies (1 mg/mL stocks) against EmVcl (1:3000), EmFAK (1:1000) and EmITGB (1:1500), in blocking solution for 1 h at RT and washed twice in 1× PBST pH 7.4. After 45 min of incubation with the secondary antibody (Alexa488^® Goat Anti-Rabbit IgG Antibody; Life Technologies, 1:1000 dilution) at RT, membranes were washed in 1× PBST pH 7.4 and imaged using the Molecular Imager FX ProPlus (BioRad).

Ephydatia muelleri juveniles were grown from gemmules for 5–7 days on No. 1.5 uncoated dishes (MatTEK) or on glass coverslips. Tissues were fixed in 4% Formaldehyde in 95% cold EtOH for 30 min–1 h at 4 °C. Juveniles were then washed three times with 1× PBS pH 7.6, and incubated in blocking buffer (3% BSA in 1× PBST pH 7.4) overnight at 4 °C. All antibody preparations were titrated to determine their optimal working concentration, from 1:250 to 1:5000. After incubation, samples were washed three times with 1× PBST and then incubated for 45 min with secondary antibody (Alexa488^® Goat Anti-Rabbit IgG Antibody; Life Technologies, 1:500 dilution), plus Alexa Fluor568^® Phalloidin (Life Technologies, 1:80) and Hoechst (33,342, 1 µg/mL) at RT. Samples were washed once in 1× PBST and twice in 1× PBS pH 7.6 and preserved for imaging using anti-fade mounting medium (0.1 M Propyl gallate, 1× PBS pH 7.6 and 90% glycerol). Confocal Images were acquired on an Olympus Fluoview FV3000 confocal laser scanning microscope using either a 20×/0.85 NA, 60×/1.4 NA or 100×/1.4 NA objectives, and processed using FIJI [69 (link)]. Neither brightness nor contrast was adjusted in the antibody channel. Immunostaining results were validated by secondary-only control and by pre-incubating each antibody with its corresponding antigen for at least 1 h at 4 °C before staining (Additional file 4: Figure S1).

Class II cytokine receptor family members, Crfbs, of zebrafish or Ginbuna crucian carp were subcloned into the p3XFLAG‐CMV™‐14 expression vector (Sigma–Aldrich, St. Louis, MO, USA) or the pcDNA6/V5‐His A vector (Invitrogen). For transient transfection, 2 × 10⁵ HEK293T cells were transfected with 1 μg of the construct DNA using the X‐tremeGENE HP Transfection Reagent (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's protocol.
Two days after transfection, HEK293T cells were incubated with 200 ng·mL⁻¹ of rIFNγrel 1 for 15 min and washed three times. The cells were then resuspended in PBS containing 0.5% FBS at a concentration of 1 × 10⁷ cells·mL⁻¹ and incubated with 1 μg·mL⁻¹ of anti‐Ginbuna IFNγrel 1 for 45 min at 4 °C. The cells were then washed three times with PBS containing 0.5% FBS, resuspended, and incubated for 30 min at 4 °C with 1 mL of a 1 : 500 dilution of Alexa 488 goat anti‐rabbit IgG antibody (Life Technologies). The cells were washed an additional three times and then suspended in 0.5 mL of PBS with 2.5 μg·mL⁻¹ propidium iodide (Life Technologies). The cells excluding dead cells were analyzed with a FACS Canto (Becton Dickinson).