Coolsnap hq2

Cells were grown on glass coverslips or on polymer µ-slide IbiTreat (Ibidi). Thereafter, cells were fixed during 10 min with methanol, permeabilized and saturated with PBS-0.2%Triton-2% BSA and hybridized with various primary antibodies (p62, vimentin, desmin, actin, HspB5, MyHC) and Alexa Fluor secondary antibodies. Hoechst 33258 reagent was used to stain nuclei (5 min, 1 ng/mL). Observations were performed on Zeiss Axio Imager Z1 photomicroscope (Zeiss Inc., Oberkochen, Germany). Images were digitized with a camera (Coolsnap HQ2; Roper scientific) and acquired with Metavue Imaging system. Digitalization was done with Metavue software; images adjustments were performed on ImageJ.

Portions (25 μl) of each elution fraction from avidin-agarose affinity chromatography were boiled in 2x Laemmli buffer and loaded to a 10% SDS-PAGE gel. Electrophoresis was carried out at 200V for 60 minutes. After completion, the gel was washed in dH₂O for 10 minutes. Then the gel was incubated with fixing solution (10% methanol, 7% acetic acid) for 1 hour at room temperature in an orbital shaker set at 50 rpm, followed by overnight incubation with SYPRO Ruby protein gel stain at room temperature with shaking and protection from light. The gel was then transferred to a clean staining container, and washed for 5 minutes with fixing solution followed by washing for 5 minutes in dH₂O.Finally, the gel was imaged in a Molecular Imager Gel Doc system (Bio Rad, Hercules) using the highest sensitivity of the CCD camera (CoolSNAP HQ2, Roper Scientific) at a resolution of 1392 x 1040 pixels with 12 bit gray scale levels per pixel.

Embryos were transferred to glass-bottom dishes (MatTek Corp., Ashland, MA) in E3 (inverted microscope) or embedded in 0.5% E3-agarose (confocal) containing 0.02% MS222. A Leica DM IRB inverted microscope (bright-field, differential interference contrast (DIC), and fluorescence imaging) coupled to a Coolsnap fx camera (Roper Scientific) was used. A Nikon AZ100 equipped for bright-field and fluorescence imaging, coupled with Coolsnap HQ2 (Roper Scientific) using MetaVue software was used to record full size embryos. Confocal microscopy was performed with an Olympus FV10i and images and movies were processed with Fluoview software and Image J. Images were processed further using Adobe Photoshop, and time-lapse videos made with image J (see specific details in movie legends). To quantify phagocytic cells, the fluorescent pixel quantification method was used as described [69 (link)]. Graphs depict macrophage and neutrophil numbers. BioImageXD [70 (link)] was used to obtain the image in S6A Fig.

Bacteria were deposited onto glass microscope slides and then covered with a thin (1 mm thick) semisolid 1% agar pad made with dilute LB (1/10 in phosphate buffer (0.05 M, pH 7.4). Images were recorded with a motorized epifluorescence microscope (Nikon TE2000-E-PFS, Nikon, France) equipped with a CoolSNAP HQ 2 camera (Roper Scientific, Roper Scientific SARL, France) and a 100x/1.4 DLL (dark low low) objective. Excitation light was emitted by a 120 W metal halide light and signal was monitored using appropriate filters. Digital analysis and image processing were conducted by a custom automation script (Visual Basic) under Metamorph 7.5 (Molecular Devices, Molecular Devices France, France), as previously described [27 (link)].

The c-Fos immunohistochemistry was performed as previously described^{53 (link)}. Briefly, anesthetized male mice were intracardially perfused with cold PBS followed by a cold 4% paraformaldehyde (PFA) in PBS. The brains were removed and post-fixed in a 4% PFA solution overnight at 4 °C. Brain regions were cut into 2–4 large blocks. The blocks were sliced on a microtome into 20-μm-thick sections. The sections were pre-incubated in blocking solution (3% bovine serum albumin and 0.3% Triton X-100 in PBS) for 1 h, then incubated with an anti-c-Fos antibody (sc-52, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the blocking solution for 12 h at 4 °C. After three washes with washing buffer, the sections were incubated with goat anti-rabbit IgG antibody coupled with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) in the blocking solution for 1 h at room temperature. The images were obtained by using an Olympus IX71 inverted microscope equipped with a cooled CCD camera (Cool SNAP HQ2; Roper Scientific, Tucson, AZ, USA). The number of c-Fos immuno-positive nuclei in each brain section were recorded and analysed using Metamorph software (Molecular Devices, Downingtown, PA, USA).

Immunohistochemistry for CD38, TRPM2, and OT were performed as described previously (Jin et al., 2007 (link)). Briefly, anesthetized mice were perfused intracardially with cold PBS followed by cold 4% paraformaldehyde (PFA) in PBS. The brains were removed and post-fixed overnight in a 4% PFA solution at 4°C. The brain regions were cut into 2–4 large blocks. The blocks were then sliced on a microtome into 20-μm-thick sections. The sections were pre-incubated in blocking solution (3% BSA and 0.3% Triton X-100 in PBS) for 1 h, and then incubated with a rabbit polyclonal antibody to mouse CD38 (sc-7049, Santa Cruz Biotechnology Inc.), a rabbit polyclonal antibody to rat TRPM2 (C-terminus) (LS-C141843, LifeSpan BioScience, Seattle, WA, USA), and a mouse monoclonal antibody to mouse OT (PS38, ATC CRL 1950) in the blocking solution for 12 h at 4°C. After three washes with washing buffer, the sections were incubated with goat anti-rabbit IgG antibody coupled with Alexa Fluor 488 (Invitrogen) in the blocking solution for 1 h at room temperature. Images were obtained using an Olympus IX71 inverted microscope equipped with a cooled CCD camera (Cool SNAP HQ2; Roper Scientific, Tucson, AZ, USA). The number of immuno-positive nuclei in each brain section were recorded and analyzed using Metamorph software (Molecular Devices, Downingtown, PA, USA).