Novolinktm polymer detection system

For immunohistochemistry, sections 3 µm in thickness were used. The primary antibodies included COX-2 (Clone SP21, Transduction Laboratories^®, Lexington, Kentucky, USA; dilution 1:40; 24 h at 4 °C) and EGFR (clone 31G7, Invitrogen^®, Paisley, Scotland, UK; dilution 1:100; 45 min at room temperature). These antibodies have been validated in canine tissues [34 (link),35 (link)].
Visualization of the primary antibodies was achieved using the NovolinkTM Polymer Detection System (Leica Biosystems^®, Newcastle, UK), with 3,3′-diaminobenzidine tetrachloride (DAB) as the chromogen, following manufacturer instructions. Subsequently, tissue sections were counterstained with Gill’s hematoxylin and cover-slipped.
The specificity of the staining was confirmed using negative controls (omitting the primary antibody) and positive controls (kidney samples for COX-2 and normal skin and mammary tumor samples for EGFR).

Independent evaluation of all renal biopsies was performed by two renal pathologists, who were blinded to data assessment and data analysis. Within each renal biopsy specimen, each glomerulus was separately evaluated for present crescents, global sclerosis, and necrosis. The percentage of glomeruli with any of these pathologies was calculated as a fraction of the total number of glomeruli in each renal biopsy. Renal biopsies were scored in accordance with the current version of the Banff score for allograft pathology, as recently described [47 (link),48 (link)]. Kidney sections (formalin-fixed, paraffin-embedded) were deparaffinized in xylene and then rehydrated in ethanol containing distilled water. Utilizing antibodies against C3c (1:10,000, A0062, Agilent Dako, Santa Clara, CA, USA) and C4d (1:50, 503-17344, Zytomed, Berlin, Germany), tissue sections were stained. According to the manufacturer’s protocol, labeling was conducted using the Novolink^TM Polymer Detection System (Leica Biosystems, Wetzlar, Germany). Nuclear counterstaining was performed using Mayer’s Hematoxylin Solution (Sigma, St. Louis, MO, USA). C3c and C4d deposits in the glomerular tuft, interlobular arteries, peritubular capillaries, and venules were evaluated for presence, as recently described [12 (link),41 (link)].

The immunostaining was performed using Novolink^TM Polymer Detection System (Leica Biosystems, UK). Following deparaffinization, rehydrated sections were boiled for 20 minutes in antigen retrieval buffer (1X Tris-EDTA, pH 9). Endogenous peroxidase activity was neutralized using peroxidase block for 5 minutes. Sections were then blocked in protein block buffer for 30 minutes and incubated with primary antibodies for 2 h at room temperature. Peroxidase chromogenic reaction was developed with DAB working solution according to manufacturer’s instructions. Slides were counterstained with haematoxylin and mounted with DPX mounting media and scanned with Hamamatsu NanoZoomer-XR Digital scanner. Staining was evaluated with Aperio ImageScope software (Leica) and the semi-quantitative H-score calculated using the following formula: $$\left[1\mathrm{x}\left(\%\mathrm{weak}\mathrm{positive}\mathrm{cells}\right)+2\mathrm{x}\left(\%\mathrm{positive}\mathrm{cells}\right)+3\mathrm{x}\left(\%\mathrm{strong}\mathrm{positive}\mathrm{cells}\right)\right].$$