Fluoromount g

Eleven LPS-treated rats, submitted to identical LTE or sham exposure protocols (n = 6 or 5 per group), were used for immunohistochemistry in the cerebral cortex. Three to four hours after exposure (or sham exposure), adult rats were deeply anaesthetized with pentobarbital and perfused with 0.1 M phosphate buffered saline (PBS) followed by paraformaldehyde 4% in 0.1 M PBS, pH 7.4. Brains were post-fixated overnight in paraformaldehyde 4%, then cryoprotected by immersion in PBS containing 25% (w/v) sucrose and frozen in − 70 °C isopentane. Immunostaining was performed on coronal sections (20 µm thick) cut on a cryostat (Microm, Heidelberg, Germany) and mounted on gelatin-coated Superfrost glass slides (Menzel-glazer, Freiburg, Germany). Sections were blocked in PBS containing 0,1% Triton-X100 and 10% goat serum (30 min at room temperature) and then incubated overnight at 4 °C with rabbit polyclonal anti-Iba1 (1:400, Wako Chemicals). Bound antibodies were detected by applying biotinylated goat anti-rabbit IgG (GE Healthcare, Velizy Villacoublay, France) for 2 h at room temperature, followed by Alexa Fluor 488-conjugated streptavidin (Life technology, Saint Aubin, France) for 30 min at room temperature. Sections were counterstained with Hoechst 33342 dye (2 µg/ml) and mounted in fluoromount G (Clinisciences). Control staining, performed by omitting primary antibodies, was negative.

Mucin 2 (Muc2) proteins were labelled on formalin-fixed samples that were cut, using a microtome (Leica RM2255, Leica, Wetzlar, Germany), into 5-μm-thick sections and mounted on adhesive microscope slides (Adhesives slides KP printer, KliniPath, Duiven, The Netherlands). Immunostaining was performed using the Leica Bond RXm. Heat-induced antigen retrieval was done with Leica Bond™ Epitope Retrieval 1 (pH6) for 20 min. Rabbit polyclonal antibody against the MUC2 protein (NBP1-31231, Novus Biologicals, Littleton, CO, USA), diluted at 1/500, was revealed by a goat anti-rabbit Alexafluor 568 IgG (dilution 1/2000, Invitrogen, Waltham, MA, USA) with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Invitrogen, Waltham, MA, USA) to counterstained nuclear. All the reagents were diluted with Bond Antibody Diluent (AR9352, Leica, Wetzlar, Germany). Slides were mounted using a fluorescent mounting medium (Fluoromount-G, Clinisciences, Nanterre, France), scanned with the Panoramic Scan 150 (3D Histech, Budapest, Hungary) and analyzed with the CaseCenter 2.9 viewer (3D Histech, Budapest, Hungary). Ten crypt (colon) or villus/crypt (ileum) units were analyzed in each sample, and the number of Muc2 cells was counted according to Fukuda et al. [17 (link)]. Tissue samples with non-well-oriented crypts were removed from the analysis.

Immunofluorescence was used to detect phospho-RPA32 (p-RPA32, serine 33 and serine 4/8), phospho-H2AX (γ-H2AX, serine 139) and 53BP1 nuclear foci. Cells treated with 5 mM hydroxyurea for 3 h were used as positive controls (Merck). Briefly, cytospins were prepared with 10⁵ cells in 1X PBS, fixed in 1X PBS/2% paraformaldehyde (Santa Cruz Biotechnologies, Santa Cruz, CA) for 20 min at room temperature and washed. Cells were permeabilized in 1X PBS/0.5% Triton X-100 for 10 min and washed in cold 1X PBS. Saturation was performed using 1X PBS/3% BSA (Euromedex, Souffelweyersheim, France) for 30 min and cells were incubated with primary antibodies for 1 h at 37 °C (Supplementary Methods 6). After washing, cells were incubated with secondary antibody for 30 min at 37 °C in the dark. DNA was counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min and cytospins were rinsed in 1X PBS and mounted with Fluoromount-G (Clinisciences, Nanterre, France). Analysis was conducted using an inverted DMI600 microscope at 100X magnification (Leica, Wetzlar, Germany). Images were analyzed using the ImageJ software (NIH, Bethesda, MD).