Abstract
BackgroundLow-vacuum scanning electron microscopy (LV-SEM) is applied to diagnostic renal pathology.MethodsTo demonstrate the usefulness of LV-SEM and to clarify the optimal conditions of pathology samples, we investigated the alterations of glomerular basement membrane (GBM) and podocytes in control and experimental active Heymann nephritis (AHN) rats by LV-SEM.ResultsOn week 15 following induction of AHN, spike formation on GBM with diffuse deposition of IgG and C3 developed. Using LV-SEM, diffuse crater-like protrusions were clearly noted three-dimensionally (3D) on surface of GBM in the same specimens of light microscopy (LM) and immunofluorescence (IF) studies only after removal coverslips or further adding periodic acid-silver methenamine (PAM) staining. These 3D ultrastructural findings of GBM surface could be detected in PAM-stained specimens by LV-SEM, although true GBM surface findings could not be obtained in acellular glomeruli, because some subepithelial deposits remained on surface of GBM. Adequate thickness was 1.5–5 μm for 10% formalin-fixed paraffin-embedded (FFPE) and 5–10 μm for the unfixed frozen sections. The foot processes and their effacement of podocytes could be observed by LV-SEM using 10%FFPE specimens with platinum blue (Pt-blue) staining or double staining of PAM and Pt-blue. These findings were obtained more large areas in 2.5% glutaraldehyde-fixed paraffin-embedded (2.5%GFPE) specimens.ConclusionOur findings suggest that LV-SEM is a useful assessment tool for evaluating the alterations of GBM and podocytes in renal pathology using routine LM and IF specimens, as well as 2.5%GFPE specimens.
Highlights
Low-vacuum scanning electron microscopy (LV-SEM) has a compact body with the advantages of high surface sensitivity and visibility, and recent studies have reported that LV-SEM is useful for ultrastructural assessments of renal pathology using light microscopy (LM) specimens [1, 2]
To examine whether LV-SEM is able to capture the ultrastructural alterations of glomerular basement membrane (GBM) and podocytes in MN, and to clarify the optimal conditions of tissue samples for LV-SEM analysis, we examined the morphological alterations of GBM and podocytes in control and experimental active Heymann nephritis (AHN) rats by LV-SEM using tissue specimens with several different fixation and staining of periodic acid-silver methenamine (PAM), platinum blue (Pt-blue), or double staining of PAM and Pt-blue
Findings of GBM in 10%formalinfixed paraffin-embedded (FFPE) sections with PAM staining by LM and LV‐SEM
Summary
Low-vacuum scanning electron microscopy (LV-SEM) has a compact body with the advantages of high surface sensitivity and visibility, and recent studies have reported that LV-SEM is useful for ultrastructural assessments of renal pathology using light microscopy (LM) specimens [1, 2]. Methods To demonstrate the usefulness of LV-SEM and to clarify the optimal conditions of pathology samples, we investigated the alterations of glomerular basement membrane (GBM) and podocytes in control and experimental active Heymann nephritis (AHN) rats by LV-SEM. Using LV-SEM, diffuse crater-like protrusions were clearly noted three-dimensionally (3D) on surface of GBM in the same specimens of light microscopy (LM) and immunofluorescence (IF) studies only after removal coverslips or further adding periodic acid-silver methenamine (PAM) staining. These 3D ultrastructural findings of GBM surface could be detected in PAM-stained specimens by LV-SEM, true GBM surface findings could not be obtained in acellular glomeruli, because some subepithelial deposits remained on surface of GBM. Conclusion Our findings suggest that LV-SEM is a useful assessment tool for evaluating the alterations of GBM and podocytes in renal pathology using routine LM and IF specimens, as well as 2.5%GFPE specimens
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