2.6 Immunohistopathological analysis
Immune cell infiltration in the kidney was determined using immunohistopathological analysis. Tissue sections were incubated with rodent declocker solution (Biocare Medical, Concord, CA, USA) at 950C for antigen retrieval (Hye Khan et al., 2016). Kidney sections were immunostained with anti-CD68 (1:100; Serotec, Raleigh, NC, USA) to determine renal macrophage/monocyte infiltration. Biotinylated rat anti-mouse secondary antibody (1:200) was used for development with avidin-biotinylated HRP complex (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA, USA) followed by hematoxylin counterstaining. Stained tissue sections were examined by light microscopy (400x magnification) and digital images were taken for analysis using Nikon NIS Elements Software. Kidney macrophage/monocyte infiltration was determined by counting CD-68 positive cells. As described earlier, (Hye Khan et al., 2016) the number of positive cells per field was divided by the metric area of the field to obtain the number of positive cells per mm2. All immunohistopathological analysis were done in blinded fashion by two observers.