Kidney Histopathology and Spleen Apoptosis Assessment

Kidney (4 mm) was fixed in 10% formalin, paraffin embedded, and stained with Periodic acid–Schiff (PAS) stain (Sigma-Aldrich) for the semi-quantitative evaluation^{21 (link),22 (link)}. Glomerular injury was determined by percentage of moderate-severe glomerular injury (mesangial expansion >50%, crescentic formation and/or glomerulosclerosis) at 400x magnification and interstitial injury was semi-quantitatively estimated at 200x magnification using 10 randomly selected fields by the criteria of damage-area (cell infiltration, interstitial edema and tubular injuries) as following: 0, <5% area; 1, 5–10% area; 2, 10–25% area; 3, 25–50% area; and 4, >50% area. The immune complex deposition in glomeruli was visualized by immunofluorescence prepared in Cryogel (Leica Biosystems, Richmond, IL, USA), stained with goat anti-mouse IgG (Alexa Fluor 488, Abcam, Cambridge, MA, USA) and detected by ZEISS LSM 800 (Carl Zeiss, Germany). Spleen apoptosis was detected by immunohistochemistry with anti-active caspase 3 antibody (Cell Signaling Technology, Beverly, MA, USA) (expressed as positive cells per high-power field)^{22 (link)} and flow cytometry. The fluorochrome-conjugated antibodies against different molecules were used including; i) apoptosis indicators, annexin V and propidium iodide (PI), ii) B220 (B cell) and iii) F4/80 (macrophage) (BioLegend, San Diego, CA, USA) with FlowJo software^{23 (link)}.