Single-molecule localisation microscopy (SMLM) is feasible in human and animal formalin fixed paraffin embedded (FFPE) tissues in medical renal disease.

Abstract

Aims: Establishment of a protocol for routine single-molecule localisation microscopy (SMLM) imaging on formalin fixed paraffin embedded (FFPE) tissue using medical renal disease including minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS).

Methods: Protocol for normal and diseased renal FFPE tissue was developed to investigate the clinical diagnostic potential of SMLM. Antibody concentrations were determined for confocal microscopy and transferred to SMLM. Different fixatives and lengths of fixation were studied. To reduce autofluorescence, additional quenching and UV bleaching steps were compared. Optimal SMLM acquisition settings were established. SMLM data were imaged, digitally captured, stored, visually inspected and analysed quantitatively.

Results: Protocol was established on normal renal FFPE tissue and then applied to clinical diseased tissue with single and multiple markers. Antibodies against key diagnostic proteins including podocin, nephrin, collagen, laminin, synaptopodin, CD31, IgG, IgM and IgA antibodies were established for MCD, FSGS and immune-mediated renal disease. We found important characteristic differences in the renal diseases listed above.

Conclusions: We established a routine super-resolution microscopy protocol for clinical FFPE material on medical renal biopsies, which could visualise fluorescently labelled proteins in all glomeruli present with a precision of approximately 10-20 nm, with a turnaround under 48 hours. We visualised and quantitated specific protein distributions in different conditions. SMLM opens subcellular microscopy in FFPE to histopathologists on routine FFPE tissue, which can in the future be an adjunct and, in some aspects, a rapid superior alternative to electron microscopy.