Fitc conjugated antibodies

Renal specimens were evaluated by direct immunofluorescence, light, and electron microscopy similar to the Huang et al. technique [26 (link)]. For direct immunofluorescence, immunoglobulins IgG, IgM, and IgA; kappa and lambda light chains; complement fractions C3 and C1q; and fibrinogen were detected by fluorescein isothiocyanate- (FITC-) conjugated antibodies (Dako, Copenhagen, Denmark) on frozen tissues. For light microscopy, paraffin sections were stained with hematoxylin and eosin, sirius red, silver methenamine stain (PAMS), and Masson's trichrome. For electron microscopy, in brief, tissue was fixed in 2.5% Karnovsky +0.2% ruthenium red and latter fixed in osmium tetroxide 2% and then dehydrated in graded alcohols and acetone solutions and embedded in Epon 812. Ultrathin sections were cut with 60 nm thickness and placed on nickel grids. Then, ultrathin sections were stained with uranyl acetate and examined with a transmission electron microscope EM-900 (Zeiss, Germany).

Renal histological lesions were graded based on the MEST-C score [17 (link)]. M0/M1 was defined as ≤ / > 50% of glomeruli exhibiting mesangial hypercellularity, E0/E1 as the absence/presence of endocapillary hypercellularity, S0/S1 as the absence/presence of segmental glomerulosclerosis, T0/T1/T2 as tubular atrophy/interstitial fibrosis ≤ 25–50% > 50%, and C0/C1/C2 as absence/ < 25%/ ≥ 25% of crescent lesions.
The immunofluorescence samples were stained with fluorescein isothiocyanate (FITC)-conjugated antibodies specific for human IgG, IgM, IgA, C3, C4, and C1q (1:50, DAKO, Glostrup,Denmark). The degree of immunofluorescence was scored on a scale of 0–4 (score 0, negative; score 1, + ; score 2, +  + ; score 3, +  +  + ; score 4, +  +  + +).