New technique of kidney tissue processing for immunofluorescence microscopy: formol sucrose/gum sucrose/paraffin

Present methods of tissue processing for immunofluorescence other than snap freezing have generally been unsatisfactory. We have developed a new kidney tissue holding and processing technique suitable for immunofluorescence, histochemical, and light microscopic analysis of the same specimen. Fifty kidney biopsies containing glomeruli in formol sucrose/gum sucrose/paraffin (FSGSP) were deparaffinized, digested with 0.1 per cent pronase, and compared both qualitatively and quantitatively with snap frozen tissue. Agreement between the two methods was very good (86 to 98 per cent), and total accuracy of FSGSP in detecting deposits ranged from 94 to 100 per cent (IgG, 94 per cent; IgM, 96 per cent; IgA, 98 per cent; fibrin, 96 per cent; Clq, 100 per cent; C3, 98 per cent). The intensity of staining based on a 0 to 4+, 11-point scale, was comparable. When both methods were positive, the intensity of staining in the same pattern was within 1 of frozen in 85 per cent of FSGSP cases and less than frozen by more than 1 in only 7.7 per cent. The overall intensity of FSGSP was slightly greater than frozen when not identical. Negative staining discrepancies were of trace amount in 73 per cent of cases with only 12 per cent differing by a quantity of 2 or greater for six stains combined. Qualitative analyses of IgE, Tamm-Horsfall protein, light chains, and alpha-2-macroglobulin were similar for the two methods. Tissue antigenicity was preserved for long periods of time in both the holding solution and paraffin. Histochemical staining for nonspecific esterase and routine light microscopic sections was comparable to regularly processed paraffin-embedded tissue. Compared with frozen methods, FSGSP was more sensitive, background was lower, few false-positives were present, resolution at high-power was better, and both linear and granular deposits were detected. This new processing method has significant advantages relative to previously described techniques and should have wide applicability to both clinical and research needs.