Sirius red fast green kit

Four μm sections of paraffin embedded kidney tissues were stained with Hematoxylin and Eosin (H&E), Periodic acid–Schiff (PAS), and Trichrome. Tissue damage was examined in a blinded manner and scored as percentage of damaged tubules: 0, no damage; 1, <25%; 2, 25–50%, 3, 50–75%, 4, >75% [15 (link)]. To evaluate renal fibrosis, the fibrotic area and fibrosis intensity in Trichrome-stained kidney sections were quantified with Image J program using published methods by an investigator blinded to the conditions [54 (link)]. To further quantify fibrillary collagen accumulation in the kidney, the kidney sections were stained with Sirius Red/Fast Green Kit (Chondrex, Inc., Redmond, WA) following the kit’s instructions [28 (link)]. Apop Tag red in situ apoptosis detection kit (EMD Millipore, MA, USA) was used for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay following the manufacturer’s protocols. Immunohistochemistry and immunoblotting were performed as previously described [55 (link),52 (link),56 (link)]. The primary antibodies used in this experiment are listed below: rabbit LC3 antibody (Novus Biologicals, CO, USA), mouse monoclonal p62/SQSTM1 antibody (Novus Biologicals, CO, USA), goat neutrophil gelatinase-associated lipocalin (NGAL) antibody (R & D, MN, USA), mouse monoclonal anti-β-actin (Sigma Aldrich, MO, USA).

Midventricular transversal sections of paraffin-embedded LV tissue (7 μm) were stained using the Sirius Red/Fast Green kit (Chondrex Inc., Redmons, WA, USA), according to the manufacturer’s instructions. After staining, sections were dehydrated in increasing concentrations of ethanol and mounted with a DPX mounting medium. Interstitial fibrosis was assessed in four randomly chosen 20× zoomed-in images using the Mirax Slide Scanner System (Carl Zeiss MicroImaging, Zaventem, Belgium). The area of collagen deposition was outlined, quantified using an automated image analysis program (Carl Zeiss, AxoVision 4.6, Zaventem, Belgium) and normalized to total surface area in %.

Transversal sections of 7 µm thick were obtained at cardiac midventricular level and stained using the Sirius Red/Fast Green kit (Chondrex). Fibrosis was assessed in all animals in four randomly chosen fields per section. The area of collagen deposition indicated by red staining was outlined and quantified using an automated image analysis program (Carl Zeiss, AxoVision 4.6, Zaventem, Belgium). Blood vessels were excluded. Total collagen deposition to the global cardiac area was calculated and expressed as percent collagen deposit.
Capillary density was quantified from histological sections by immunohistochemical staining for CD31 (SC-1506, Santa Cruz, 1/100). Capillaries were visualized by 3-3-diaminobezedine (DAB) and counterstained using hematoxylin. The amount of blood vessels were counted in 10 different fields per section and averaged. Data are expressed as amount of capillaries per µm².