Sirius red f3ba

Tissue samples from at least two representative fragments of each liver lobe were taken at necropsy, fixed in 10% phosphate-buffered formalin for 12–24 hr, and then embedded in paraffin. Serial 4-μm sections were stained with H&E to histologically determine the number and size of nodules. For Sirius Red staining, 4-μm liver tissue sections were deparaffinized, rehydrated, and then stained for 1 hr in saturated picric acid with 0.1 Sirius Red F3BA (Aldrich Chemicals, St. Louis, MO, USA) at room temperature. Next, the slides were washed twice with acetic acid solution and finally dehydrated in a graded alcohol series. Sections were evaluated using an image cytometer consisting of a single 2/3-in charge-coupled device (CCD) color camera (JVC Professional Europe, London, UK) mounted on a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany) equipped with a motorized scanning table (Märzhäuser, Wetzlar, Germany) controlled by Cytometrica software (C&V, Bologna, Italy). The fibrotic area was quantified based on four different fields (acquired at low magnification, 2.5× ) for each slide using ImageJ Software (https://imagej.nih.gov) and expressed according to the following formula: [collagen area/(total area − vascular lumen area)] × 100.

To asses renal collagen content, paraffin-embedded kidneys were cut into 4-mm slices and stained with Sirius Red F3BA (0.5% in saturated aqueous picric acid; Aldrich Chemical Company, Madrid, Spain). Four different sections of each slide of the kidney and ten photographs from each section were taken using an image analysis system (Leica Microsystems, Barcelona, Spain). A single investigator, blinded to the nature of the samples, performed the analyses.

Picrosirius red staining was performed and analyzed as previously described [23 (link)]. Briefly, tissues were deparaffinized, hydrated and stained with 0.1% Sirius red F3BA in saturated aqueous picric acid (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Nuclei were counterstained with Weigert’s hematoxylin (Sigma-Aldrich, St. Louis, MO, USA), dehydrated, and mounted. Birefringence was captured under circular polarized light and the density of 4 different colors (green, yellow, orange, red, indicative of elevating collagen bundle thickness) was quantified. The ratio of individual colors was expressed relative to the number of total positive pixels. Total collagen was calculated as the proportion of positive pixels relative to the tissue area calculated in ImageJ with thresholding of a bright field image captured at the same position as with the polarized light.

Paraffin-embedded kidney tissues were stained with Sirius red F3BA and Food green FCF (Sigma Aldrich) overnight. After washing with PBS buffer, the dye was eluted from tissue sections with 0.1 N sodium hydroxide methanol. Absorbance at 540 and 605 nm was determined for Sirius red F3BA and Food green FCF-binding protein, respectively. This assay provides a simple, relative measurement of the ratio of collagen to total protein, which is expressed as micrograms per milligram of total protein.

For Sirius red staining, to specifically stain fibrillary collagen, slides were deparaffinised and immersed for 30 min in saturated aqueous picric acid containing 0.1% Sirius red F3BA (Sigma-Aldrich, Milan, Italy) [64 (link)]. The slides were photographed with a digital camera connected to a Nikon Eclipse 80i microscope. Collagen content was quantified blind by three different operators by image analysis in at least 8 random fields and expressed as fibrosis index, that is, the ratio between the collagen relative to the whole section area analysed, expressed as a %.
On the same sections analysed for collagen content, the number of centronucleated skeletal muscle fibres was quantified in order to analyse the eventual relationship between skeletal muscle injury and fibrosis.