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.