Qwin 500 c image analyzer

Kidney tissues were fixed in formal saline (10%) and paraffin blocks were prepared using standard procedure. Sections of 5 μm thickness were stained with hematoxylin and eosin (H&E) for basic staining histological analysis [44 (link)]. To quantify the deposition of collagen fibers in kidney tissues, Sirius red staining was performed. Following dewaxing and rehydration of kidney tissue sections, slides were incubated with 0.1% Sirius red (Sigma-Aldrich, Gillingham, UK) overnight, dipped in 0.01 M hydrochloric acid, and dehydrated with serial ethanol concentrations without water. Determination of collagen deposition (area percentage) in sections stained with Sirius red and Bowman’s capsule space (μm) was evaluated in 10 nonoverlapping fields for each group using a Leica Qwin 500 C image analyzer (Cambridge, UK). Data were summarized as means ± SD and compared using ANOVAs followed by a Tukey test. p-values < 0.05 were considered statistically significant. Calculations were made using SPSS software, version 19.

Immunohistochemistry for the paraffin sections was carried out using rabbit anti-caspase 3 (dilution 1:500; Cat. No. NB100–56113, Novus Biologicals, Littleton, CO, USA) as a marker for apoptosis [64 (link)], rabbit anti-TNF-α (dilution 1:200; Cat. No. NB600–587, Novus Biologicals, Littleton, CO, USA) and mouse anti-interleukin-6 (IL-6) (dilution 1:400; Cat. No. ab9324, Abcam, Cambridge, UK) as markers of inflammation [47 (link)]. Antigen retrieval was performed by heating in citrate buffer then quenching of endogenous peroxidase was processed using a peroxidase blocker. The sections were incubated with the primary antibodies (Ab) in a humidity chamber overnight followed by incubation with the secondary Ab for 45 min. Visualization of the reaction was performed using a 3,3′-Diaminobenzidine (DAB) detection kit with counterstain by Meyer hematoxylin. The area % of the (+ve) immunostaining for caspase3, TNF-α and IL-6 was measured using Leica Qwin 500 C image analyzer. The measures were obtained from 10 non-overlapping low power fields/section in each group.