Immunohistochemistry and immunofluorescence
Immunohistochemical staining was performed on 3-μm paraffinized sections
of renal tissue. Detection of CD68, CD4, CD8 and CD20 (Maxim-Bio),
tryptase and CD1c (Abcam) was performed on paraffin sections by an
immunohistochemical kit (DAKO, Santa Clara, USA) according to the
manufacturer’s instructions. For immunofluorescence, the primary
antibody against C3, IgA, IgG, and IgM (Gene-Bio) and Alexa Flour
488-conjugated secondary antibody (Jackson ImmunoRearch, West Grove,
USA) were used. The numbers of each subset of interstitial inflammatory
cells were counted under five equivalent high-power cortical fields (HPFs)
(×400) and were expressed as the average number of cells per square
millimeter [16]. The labeled cells were counted three times per high
power field (HDF) to reduce counting errors, and the average number was
recorded as the density of positive cells in this renal section.