N. P. Goode

Detection of glomerular anionic sites in post-embedded ultra-thin sections using cationic colloidal gold.

We detected glomerular anionic sites in fixed, LR Gold-embedded ultra-thin tissue sections using cationic colloidal gold. Manual and computer-assisted quantitation were compared, and the influence of pH and glycosaminoglycan-degrading enzymes on site expression was examined. Both quantitation methods produced similar results. Alteration of pH within a narrow range (pH 2.5-3.0) markedly affected the staining pattern. At pH 2.5, epithelial and endothelial glycocalyx and regular sites restricted to the lamina rara externa were stained. At pH 3.0 and above, glycocalyx was unstained but intracellular and nuclear staining was present; glomerular basement membrane (GBM) and mesangial matrix sites were abundant. After chondroitinase ABC or hyaluronidase digestion, GBM staining was eliminated at pH 2.0 and reduced at pH 7.0 (p less than 0.001), suggesting that degraded sites are associated with chondroitin sulfate or hyaluronic acid. By contrast, prolonged heparitinase I digestion was ineffective at either pH. Digestion of purified substrates revealed crossreactivity of heparitinase towards chondroitin sulfate and of chondroitinase towards hyaluronic acid. Since tissue sites were reduced by chondroitinase but not heparitinase, we suggest that degradation is due to hyaluronidase activity of chondroitinase and the anionic sites are associated with hyaluronic acid. However, the influence of pH indicates that lamina rara externa sites are structurally distinct from other GBM anionic sites.

Expression of an Antigen Associated with Basal Bodies of Human Ciliated Epithelial Cells

The process and regulation of ciliogenesis in human epithelia is little understood and many components of the cilium and associated structures have not been characterised. We have identified a monoclonal antibody, LhS28, which recognises a 44,000–45,000 Mr protein specifically associated with human ciliated epithelial cells. Immunoperoxidase labelling of formalin-fixed paraffin wax-embedded human tissues showed that LhS28 was expressed in the sub-apical zone of ciliated epithelial cells of the Fallopian tube and upper respiratory tract, but not ciliated ependyma, non-ciliated epithelia or testis containing developing spermatozoa. Immunoelectron microscopy demonstrated that the antigen recognised by LhS28 was associated with the basal body structure of the cilium and specifically with the 9+0 microtubule arrays. LhS28 should be a useful tool in the identification of ciliated cells in pathological specimens and for investigating mechanisms of ciliogenesis.

Spontaneous glomerular immunoglobulin deposition in young Sprague-Dawley rats

The frequency, age-onset and distribution of spontaneously deposited immunoglobulins (lgs) in glomeruli of Sprague-Dawley rats has been investigated. Groups of rats (n=10) were examined at 4-7 day intervals from birth (presuckling) until 30 days of age. Findings were compared with circulating immunoglobulin concentrations in each age group. Immunoglobulins were undetectable in immature kidneys of newborn rats. However, as early as 5 days, scanty IgA and IgM deposits were observed predominantly in mesangial areas of mature glomeruli, corresponding to low circulating concentrations of these immunoglobulins. By contrast, glomerular IgG deposits were not observed until 21 days, despite relatively high concentrations of circulating maternal IgG from birth. Mesangial deposition of immunoglobulins increased with age. Absence of complement C3c or electron dense deposits associated with this mesangial localization suggests that immunoglobulins were not deposited as immune complexes. Accumulation of non-phlogogenic immunoglobulins in the mesangium of normal rats supports the concept that the mesangium is constantly perfused by circulating macro-molecules and filtration residues. The results indicate problems of interpretation of the significance of endogenous immunoglobulin deposition in models of experimental glomerulonephritis, even in studies involving weanling rats.

Preparation, purification and analysis of BSA-coated latex particles for in vivo studies: non-ionic adsorbance of unbound protein ligand

A procedure for the preparation, purification and analysis of small, BSA-coated carboxylate-modified latex (CML) particles, suitable for use in in vivo studies has been developed. Following conjugation, uptake of unbound BSA by Amberlite XAD-8 non-ionic adsorbent beads has been shown to be an effective method by which unbound protein ligands may be removed from coated latex preparations.

Immunogold-silver staining of mesangial antigen in lowicryl K4M- and LR gold-embedded renal tissue using epipolarization microscopy.

We examined the localization of a glomerular mesangial antigen with a Thy-1.1 monoclonal antibody by epipolarization microscopy (EPI) of silver-enhanced, immunogold-stained renal tissue embedded in LR Gold and Lowicryl K4M, and compared the attributes of these hydrophilic resins. Antigen was well preserved in tissue embedded in both resins. LR Gold-embedded tissue demonstrated excellent immunostaining properties, sectioned more easily, and showed better durability during staining than K4M. Lowicryl K4M-embedded tissue, however, displayed a phenomenon of self-illumination when counterstained with eosin which was not seen with LR Gold. This enabled immunostaining to be precisely related to tissue morphology without the necessity of simultaneous transillumination, which can be problematic when used in combination with EPI because of reflection of incident illumination from sub-stage optical surfaces.

Cationic gold staining of glomerular anionic sites in archived tissue, reprocessed from paraffin wax into LR gold resin

SummaryGlomerular capillary wall anionic sites have been demonstrated by cationic gold staining of archived renal biopsy tissue (up to 10 years old), obtained from six patients, originally embedded in paraffin wax, and subsequently reprocessed into LR gold resin. The staining patterns at pH 2.5 and pH 7.0, demonstrating different glomerular basement membrane (GBM) anionic constituents, were compared in three patients from whom tissue directly processed into LR gold and reprocessed tissue was available. Ultrastructural preservation was poorer and shrinkage artefact greater in paraformaldehyde-lysine periodate (PLP) as opposed to formol saline-fixed reprocessed tissue. However, GBM anionic site expression was well preserved, or even enhanced (lamina rara externa, pH 7.0) in reprocessed tissue, using either fixative. Although it may not be possible to compare subtle changes in anionic site distribution in variously fixed and processed tissues, due to these artefacts, the technique enables retrospective study of charge status in archived material from disease groups in which there are distinct anionic site aberrations.