4.7. Immunohistochemistry and Morphometric Analyses

Methacarn-fixed kidneys were embedded in paraffin, sectioned (3 µm), and stained using the avidin-biotin complex (ABC) method, as previously described [11,20]. Antigen retrieval was done by steam-heating using a decloaking chamber pro (Biocare Medical, Concord, CA, USA). Sections were incubated with the primary antibodies overnight at 4 °C and with the biotin-conjugated secondary antibodies for one hour at room temperature. The following primary antibodies and dilutions were used: mouse anti-rat Ki-67 (M7248, Dako; 1:25), mouse anti-rat α-smooth muscle actin (α-SMA; A2547, Sigma; 1:200), mouse anti-rat CD68 (MCA3476A9, Bio-Rad; 1:500), rabbit anti-rat CD3 (ab5690, abcam; 1:100) and mouse anti-rat Desmin (M0760, Dako; 1:100). Anti-mouse (1:500) and anti-rabbit (1:300) secondary antibodies were from Jackson ImmunoLab (715-065-151 and 711-065-152, respectively). For each specific staining, a mouse or rabbit isotype (IgG) condition (in place of the primary antibody) was used as a negative control. Immunostained sections were counterstained with hematoxylin.

Morphometry of immunostained sections was performed by an observer blinded to their origin. For each slide, pictures of 20 randomly selected glomeruli were taken using a Zeiss digital camera (Axiocam ICc1 and AxioVision Software) on a Axiostar Plus microscope (Carl Zeiss, Jena, Germany). CD68-, CD3- and Ki-67-positive cells were enumerated using the HistoQuest software (TissueGnostics GmbH, Vienna, Austria) and respective arithmetic means (±SD) were calculated. Ki-67-positive cell counts were expressed as a percentage of the total number of glomerular cells. The scoring system for CD3-positive cells (0 to 3) was extended to evaluate the interstitial kidney compartment, including the periglomerular, -arterial, -venous, and -tubular areas. CD3 staining was scored as follows: no staining (score 0), isolated stained cells (score 1), multiple clusters of stained cells (score 2), and strong or continuous staining around structures (score 3). α-SMA staining in mesangium was scored (0 to 4) according to Johnson et al. [21], as follows: no staining in glomerulus (score 0), segmental staining in the mesangial area (score 1), staining outlines the mesangial stalk (score 2), staining of the mesangial stalk with focal areas of nodularity (score 3), and staining of the entire glomerulus with diffuse areas of nodularity (score 4). Desmin staining of the outer edge of the glomerular tuft was scored (0 to 4) according to the percentage of outer edge with positive staining [22], as follows: 0 to 5% stained (score 0), 5% to 25% stained (score 1), 25% to 50% stained (score 2), 50% to 75% stained (score 3), and ≥75% stained (score 4).