Cellsens dimension imaging software version 1

Histological sections, 5 μm thick, were cut from the paraffin-embedded posterior and anterior eye samples. The sections were deparaffinized and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0; Bio-Optica, Milan, Italy) for antigen retrieval. After a pre-incubation in 1.5% bovine serum albumin (BSA) in PBS, pH 7.4 for 20 min at RT to minimize the non-specific binding, the sections were incubated overnight at 4 °C with primary antibodies directed against H1-4, diluted 1:100 in 5% BSA PBS-T (Santa Cruz Biotechnology, Dallas, TX, USA), followed by secondary Alexa Fluor 594-conjugated IgG (1:500; Jackson ImmunoResearch Europe Ltd., Cambridge House, UK).
The sections were mounted in an aqueous medium (Fluoremount, Sigma, Milan, Italy) with 4′,6-diamidino-2-phenylindole (DAPI). Representative images were acquired by an Olympus BX63 microscope coupled to CellSens Dimension Imaging Software version 1.6 (Olympus, Milan, Italy).

Histological sections, 5 µm thick, were cut from the paraffin-embedded posterior and anterior eye samples, deparaffinized and incubated with fluorescent DHE 2µM (Abcam, Camdridge, UK) in a humidity chamber for 30 min at 37 °C. Coverslips were then placed on the slides and kept in the dark. The sections were mounted in an aqueous medium (Flouremont, Sigma, Milan, Italy). Flourescence was detected (absorbance 518 nm, emission 605 nm) using an Olympus BX61 microscope coupled to CellSens Dimension Imaging Software, version 1.6 (Olympus, Milan, Italy)

Immunofluorescence analysis was performed as previously described (Lucarini et al., 2014 (link)). Briefly, histological sections of 5 μm thick were deparaffinized and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0, Bio-Optica, Milan, Italy) for antigen retrieval. A pre-incubation in 1.5% bovine serum albumin (BSA) in PBS, pH 7.4 for 20 min at RT was necessary to minimize the unspecific binding; whereupon, the sections were incubated overnight at 4°C with rabbit monoclonal anti-αSMA antibody (1:200 ABCAM, USA) followed by goat anti-rabbit Alexa Fluor 488-conjugated IgG (1:300 Invitrogen, San Diego, CA, USA) for 2 h in the dark at RT. Negative controls were performed with non-immune rabbit serum substituted for the primary antibody. The counterstaining of nuclei was obtained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images were acquired with an Olympus BX63 microscope coupled to CellSens Dimension Imaging Software version 1.6 (Olympus, Milan, Italy). αSMA expression was quantified by densitometric analysis of fluorescence signal intensity, measured on digitized images using ImageJ software². Twenty regions of interest (ROI) were evaluated for each sample. Values are expressed as mean ± SEM of the OD measurements (arbitrary units) of individual mouse from the different experimental groups.