Dual detection of TNFa mRNA and protein

A tnfa probe was amplified from total cDNA by PCR using tnfa.55 and tnfa.58 primers (Supplementary file 1) and cloned in plasmid pCRII-TOPO. Digoxigenin (DIG)-labelled (Roche, France) sense and anti-sense RNA probes were in vitro transcribed (Biolabs, France). In situ hybridizations on whole-mount embryos were as previously described (Nguyen-Chi et al., 2012 (link)). For simultaneous detection of eGFP-F proteins and tnfa mRNA by immuno-detection and in situ hybridization, fixed and rehydrated Tg(tnfa:eGFP-F) larvae were permeabilised in ice in 100% ethanol for 5 min, then in a mixture of 50% Xylene-50% ethanol for 1 hr and in 80% acetone for 10 min at −20°C as described in Nagaso et al. (2001) (link). After washes in PBS-0.1% Tween, larvae were post-fixed in 4% paraformaldehyde (PFA) for 20 min. Subsequent steps of hybridization, washes, and staining with NBT-BCIP (Roche, France) were as previously described in Nguyen-Chi et al. (2012) (link). Next, unspecific-binding sites were saturated in PBS-1% bovin serum albumin (BSA)-1% lamb serum-10% Goat serum and larvae incubated 3 days with an anti-GFP antibody (MBL, 1/500). After extensive washes, larvae were incubated with a goat anti-rabbit antibody. Stained embryos were imaged using a MVX10 Olympus microscope with MVPLAPO 1× objective and XC50 camera and using a Zeiss Axioimager with a Zeiss 40× Plan-Apo 1.3 oil objective.