Sp5 2 confocal laser scanning microscope

Embryos were laid overnight at 23°C on apple juice agar dishes, dechorinated in bleach and mounted on Greiner Lumox gas-permeable culture dishes (Sigma) in halocarbon oil 700 (Sigma) or on glass slides with double-sided tape with Voltalef oil (VWR International). For experiments using RNAi, embryos were laid at 28°C. Embryos were wounded at stage 14 using a nitrogen ablation laser (Spectra-Physics) attached to a Zeiss Axioplan 2 widefield imaging system. Confocal imaging was performed on a Leica SP5-II confocal laser scanning microscope. Image preparation and analysis was performed on maximum projected confocal movies and utilised Volocity (PerkinElmer) and ImageJ (NIH) software. Graph plotting and statistical analysis were performed using Prism (Graphpad). Image quantification methods can be found in the supplementary material.

HeLa cells were grown to 70% confluence on poly-lysine coverslips. Cells were infected with log phase Shigella at a MOI of 20. An Afa1-expressing bacteria strain was used to increase bacterial adhesion61 (link). The samples were centrifuged for 10 min at 900g at room temperature to synchronise adhesion, then incubated for 6 min at 37 °C to initiate interaction with host cells. Cells were fixed with 2% PFA, blocked with 3% w/v BSA in PBS and then immunostained for 1 h at room temperature with anti-IpaD, anti-IpaH and anti-MxiH (see FACS section for santibody details). Host cell actin was stained with Alexa Fluor 488 Phalloidin (Life Technology) according to the manufacturer’s instructions. Samples were mounted with Mowiol. Images were taken using a Leica SP5-II confocal laser scanning microscope using a 63X oil objective.

All experiments were carried out according to UK Home Office regulations. Lines used were Tg(col2a1BAC:mCherry) (Hammond and Schulte-Merker, 2009 (link)), Tg(Fli1:eGFP)^y1 (Lawson and Weinstein, 2002 (link)) and Sox10:eGFP (Wada et al., 2005 (link)). Morpholinos (Gene Tools) against coronin-1ca (ENSEMBL gene ID: ENSDARG00000035598) (translation blocking, 5′-TCGTACAACCCGTTTGAACATATCT-3′, 3.5 nM) and RCC2 (ENSEMBL gene ID: ENSDARG00000011510) (splice blocking, 5′-ATACAAGAAGCATCCTTACAATCTT-3′, 1 ng) were injected at the one-cell stage. For rescue experiments, 200 pg human mRNA, prepared using a mMessage mMachine kit (Ambion), were co-injected with the morpholino. Images were captured on a Leica SP5-II confocal laser-scanning microscope. Alcian Blue and Alizarin Red staining is as previously described (Walker and Kimmel, 2007 (link)).

Wandering third instar larvae were dissected in ice-cold, Ca^2 +-free HL3.1-like solution (in mM: 70 NaCl, 5 KCl, 10 NaHCO₃, 115 sucrose, 5 trehalose, 5 HEPES, 10 MgCl₂) to produce a larval “fillet” (Brent et al., 2009 ). The fillet was fixed for 30 min in 4% paraformaldehyde (Sigma Labs) and washed three times in 1% Triton-X (Sigma Labs), then blocked for one hour in 5% normal goat serum (Fitzgerald Industries) and 1% Triton-X at room temperature. It was incubated overnight in 1:500 FITC-conjugated anti-horseradish peroxidase (HRP-FITC) (Jackson Immunoresearch Laboratories) and 1:500 mouse anti-Discs large (Dlg) primary antibody (Sherwood et al., 2004 ), then for two hours in 1:500 AlexaFluor 633-conjugated goat anti-mouse secondary antibody at room temperature. Fillets were washed and mounted on a coverslip in Vectashield (Vector Laboratories). Z-series of larval NMJs were imaged on a Leica SP5-II confocal laser-scanning microscope using an oil immersion 40 × objective. The number of boutons at the NMJ of muscle 6/7 in segment A2 was counted manually. Satellite boutons were identified as a single bouton with 3 or more boutons budding from it (Menon et al., 2013 (link)). ImageJ (rsb.info.nih.gov/ij/) was used to manually outline muscles 6 and 7 and hence calculate their area.

Wandering third instar larvae were dissected in ice-cold, Ca²⁺-free HL3.1-like solution (in mM: 70 NaCl, 5 KCl, 10 NaHCO₃, 115 sucrose, 5 trehalose, 5 HEPES, 10 MgCl₂) to produce a larval “fillet” (Brent et al., 2009 ). The fillet was fixed for 30 min in 4% paraformaldehyde (Sigma Labs), washed three times in 1% Triton-X (Sigma Labs) and blocked for one hour in 5% normal goat serum (Fitzgerald Industries) and 1% Triton-X at room temperature. It was incubated overnight in 1/500 mouse FITC-conjugated anti-horseradish peroxidase (HRP-FITC) (Jackson Immunoresearch Laboratories, 115–035-003) and 1/500 rabbit anti-Discs large (Dlg) (Biocompare, ABIN1387516) primary antibody, then for two hours in 1/500 AlexaFluor 633-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific, A-21052) at room temperature. Each fillet was washed and mounted on a coverslip in Vectashield (Vector Laboratories). Z-series of NMJs were imaged on a Leica SP5-II confocal laser-scanning microscope using an oil immersion 40 × objective. The number of boutons at the NMJ of muscle 6/7 in segment A2 was counted manually. ImageJ (rsb.info.nih.gov/ij/) was used to manually outline muscle 6 and hence calculate their area.

Overexpression of mnb was driven in the eye using the Glass multimer reporter (GMR-Gal4). Images of the whole head of 1–2 day old flies were taken via a Zeiss AxioCam MRm camera attached to a stereomicroscope (Zeiss SteREO Discovery.V8, up to 8× magnification), and the surface area of the eye was calculated by manually outlining the eye in ImageJ (rsb.info.nih.gov/ij/). Overexpression of mnb in GFP-tagged ellipsoid body (EB) ring neurons was achieved by crossing EB1-Gal4; UAS-mCD8-GFP flies with UAS-mnb flies. Following published methods (Williamson and Hiesinger, 2010 ), adult brains were dissected, fixed for 30 min in 4% paraformaldehyde and mounted on a coverslip in Vectashield (Vector Laboratories). Slides were imaged on a Leica SP5-II confocal laser scanning microscope using an oil immersion 40 × objective. A Z-stack of 25 images at 1 μm increments was captured and combined into a 3-D projection using ImageJ (rsb.info.nih.gov/ij/); analysis was performed by scrolling through all 25 images and counting the number of cells in one brain hemisphere.