Nbt bcip bm purple

In situ hybridization was performed using DIG-labeled RNA probes and anti-DIG::AP antibody (Roche). Signal was developed using NBT/BCIP (BM Purple, Roche), or Fast Red/HNPP (Roche). Immunocytochemistry was performed using anti-Eve (mouse monoclonal antibody 2B8, hybridoma bank, University of Iowa) as primary, and anti-mouse::POD as secondary antibody (ABC kit, Vector). AlexaFluor 488 tyramide (Invitrogen) was used to give green fluorescent signal. All expression analyses were performed using embryos from uninjected GA-1 strain (WT) or adult GA-1 females injected with double-stranded RNA (ds RNA) of the gene of interest. dsRNA was synthesized using the T7 megascript kit (Ambion) and mixed with injection buffer (5 mM KCl, 0.1 mM KPO₄, pH 6.8) before injection. Used dsRNA concentrations: 200 ng/µl for severe Tc-cad, 7.5 ng/µl for mild Tc-cad, 200 ng/µl for Tc-lgs, 200 ng/µl for Tc-pan, 1 µg/µl for Tc-apc1, 1 µg/µl for Tc-zen1, and 200 ng/µl; 1 µg/µl for Tc-lgs;Tc-zen double RNAi.

In situ hybridization was performed using DIG-labeled RNA probes and anti-DIG::AP antibody (Roche). Signal was developed using NBT/BCIP (BM Purple, Roche) according to standard protocols (Schinko et al., 2009 (link); Shippy et al., 2009 (link)). All expression analyses were performed using embryos from uninjected females or females injected with double-stranded RNA (dsRNA) of gene of interest. dsRNA was synthesized using the T7 megascript kit (Ambion) and mixed with injection buffer (5 mM KCl, 0.1 mM KPO₄, pH 6.8) before injection. Used dsRNA concentration for axn RNAi: 100 ng/µl. Embryos were imaged using ProgRes CFcool camera on Zeiss Axio Scope.A1 microscope and ProgRes CapturePro image acquisition software. Brightness and contrast of all images were adjusted and placed on a white background using Adobe Photoshop.