Sodium citrate buffer

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).

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.

Immunofluorescence staining was performed as previously reported [3 (link)]. Briefly, lung sections were deparaffinized and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0, purchased from Bio-Optica, Milan, Italy) for antigen retrieval. Successively, the sections were immune-stained with rabbit monoclonal anti-α-SMA antibody (1:200; Abcam, Cambridge, UK) or rabbit monoclonal anti-NF-kB p65 antibody (1:400, Cell Signaling Technology, Danvers, MA, USA) and goat anti-rabbit Alexa Fluor 568-conjugated IgG (1:300; Invitrogen, San Diego, CA, USA). The negative control was the section in which non-immune rabbit serum was substituted for the primary antibodies. DAPI was used as counterstaining, and representative images were acquired by an Olympus BX63 microscope (Milan, Italy) equipped with Olympus CellSens Dimension Imaging Software version 1.6. The markers’ expression was quantified by densitometric analysis of the intensity of the fluorescence signal in digitized images with ImageJ software. Values are expressed as mean ± SEM of the OD measurements (arbitrary units) of individual mice (20 images each) from the various experimental groups.