Vectorshield with dapi

Fluorescein isothiocyanate (FITC)–conjugated rat anti–mouse CD11b (1:100; monocyte/macrophage marker; Biolegend, CA) and FITC-conjugated rat IgG2bk (isotype control; Biolegend) were used for the immunohistochemical assay. Three corneas from three mice per group were used and dissected corneas were fixed with acetone for 15 min at Day 14, as previously described [22 (link),23 (link)]. After blocking nonspecific staining with an anti-FcR CD16/CD32 antibody (Biolegend), the specimens were immunostained with primary or isotype antibodies overnight, washed three times with PBS, incubated with secondary antibodies, and mounted with Vector Shield with DAPI (4,6 diamidino-2-phenylindole, Vector Laboratories). CD11b⁺ cells were counted at two areas in the center (within 2 μm of the center) of each cornea in a masked fashion under an epifluorescence microscope (Nikon E800) at 40× magnification. The mean number of cells was analyzed by averaging the cell number in each area. The data are presented as averages ± SEM of all mice observed.

Cells were grown on cover slips overnight, then fixed in 4% paraformaldehyde at room temperature for 20 min, washed with phosphate buffered saline (PBS), and permeabilized with 0.5% Triton X-100 PBS for 10 min. After blocking for 1 h in 5% BSA in PBS, cells were incubated with EGFR (1:200, sc-120, Santa Cruz Biotechnology) antibody diluted in 5% BSA/PBS for 1 h at room temperature, followed by incubation with Alexa Fluor 568 Donkey Anti-Rabbit IgG antibody (A11034, Life Technologies, Carlsbad, CA) for another 1 h at room temperature. Cells were washed with PBS and mounted in Vector shield with DAPI (Vector Laboratories, Burlingame, CA). Finally, images were taken by using a LSM710 confocal microscope (Carl Zeiss, Jena, Germany) at ×200 magnification.

Cells were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and blocked with 4% BSA. The cells were then incubated with primary antibody in 1% Triton X-100 at 4 °C overnight, followed by incubation with secondary antibody in 1% Triton X-100 for 1 h at room temperature. Coverslips were mounted using Vectorshield with DAPI (Vector Laboratories, Burlingame, CA, USA) and visualized on AxioObserver Z1 Microscope (Zeiss, Oberkochen, Germany) with Plan-Apochromat × 40 1.25 objective. Images were captured using a Roper Scientific CoolSNAP CCD camera.

Primary WT, primary CIZ1 null and culture adapted WT murine embryonic fibroblast cells were grown on glass coverslips and washed with 0.1% Triton-X-100 containing PBS prior to fixation with 4% paraformaldehyde for 15 min. Coverslips were blocked and incubated with primary antibodies as usual before washing with Duolink^® In Situ Wash Buffer A and the proximity ligation assay detailed in Duolink^® PLA fluorescence protocol (Sigma), using species-specific secondary antibody probes and green detection reagents. In brief, following standard primary antibody incubation as detailed in antibodies and detection protocols, coverslips were washed twice for 5 min with Wash Buffer A and incubated with species-specific PLUS and MINUS PLA probes in the provided antibody diluent for 1 h at 37 °C. Coverslips were washed twice for 5 min with Wash Buffer A before incubation for 30 min at 37 °C with 1× Ligation Buffer supplemented with DNA Ligase. Coverslips were washed twice for 5 min with Wash Buffer A followed by incubation for 100 min at 37 °C in Amplification buffer supplemented with DNA polymerase. Coverslips were washed twice for 10 min with Wash Buffer B before a final 1 min wash in 0.01× Wash Buffer B and mounting in Vector Shield with DAPI (Vector Labs).

5-Ethynyl-2ʹ-deoxyuridine (EdU, 10 μM) was added to adherent cells on coverslips at ~70% confluence for a 30 min pulse period under standard growth conditions. For pulse/chase experiments, coverslips were transferred to warm PBS then into fresh media (without EdU) for 30–60 min. To visualize newly synthesized DNA, coverslips were washed briefly in CSK with 0.1% Triton-X-100, or subjected to extraction up to the RNase treatment step before fixation with 4% paraformaldehyde for 15 min. Coverslips were then washed in PBS and EdU detected using the Click-iT® EdU Alexa Fluor® 594 kit (ThermoFisher), as recommended. Briefly, coverslips were blocked with 3% BSA before incubation in a light-proof humidified chamber with Click-iT® reaction cocktail for 60 min. For dual staining (e.g. CIZ1 or fibrillarin), coverslips were first incubated with primary antibody as described under antibodies and detection protocols before blocking with 3% BSA. Anti-species secondary antibody diluted in Click-iT® reaction buffer was included in the EdU detection step. Coverslips were then washed and mounted using VectorShield with DAPI (Vector Labs).

10-µm heart cryo-sections were mounted on poly-L-lysine-coated slides and fixed with 4% paraformaldehyde for 20 min, washed with PBS, incubated with rhodamine-linked wheat germ agglutinin (WGA; 1:1000; Vector Laboratories, UK) for 2 h, washed again with PBS and mounted in VectorShield with DAPI (Vector Laboratories, UK). To assess macrophage infiltration, fixed heart cryo-sections were permeabilized with 0.5% Triton X-100 for 20 min, then blocked with 10% horse serum in PBS for 1 h prior to incubation in 10% horse serum/PBS containing 1/100 rat anti-CD68 antibody (Abcam, UK) and 1/1000 WGA for 2 h. The sections were rinsed twice with PBS, incubated with 1/1000 anti-rat IgG-FITC secondary antibody (Abcam, UK) in 10% horse serum/PBS for 1 h, washed three further times with PBS and mounted in VectorShield with DAPI. High-resolution thin-section (0.5 µm) images were taken by confocal microscopy using a Zeiss LSM Axiovert 200M microscope fitted with a 63× water objective, Zeiss LSM510 software and Adobe Photoshop CS. Images in Fig. 1 were taken using a Zeiss AxioImager epifluorescence microscope with AxioVision digital image processing software. CM cross-sectional area counts were averaged from >four animals per group, five fields of view per animal (40× objective) from healthy areas of the left ventricle.