The use of post‐embedding immunoelectron microscopy in the diagnosis of glomerular diseases

Skjørten

et al. 1992

APMIS

A previous light microscopic study on cryogenic brain lesions in rats demonstrated uptake of plasma proteins into damaged neurons within a few minutes after the lesion. The protein concentration was much higher inside the nerve cell bodies than in the surrounding neuropil. This is puzzling since the neuropil to a large extent consists of damaged neuronal processes. The present investigation describes the intracellular localization of albumin in this model using a post-embedding immunoelectron microscopic technique. The distribution of albumin in the lesions was studied after 1, 6 and 12 h survival periods. The intraneuronal albumin was mainly bound to the particulate elements of the cytoplasm and nuclei, while the watery parts of the cells showed no immunoreactivity. The intracellular organelles contained very little albumin, indicating that their membranes may be more resistant to freezing than those of the cells. Most of the neuronal and glial processes in the neuropil were swollen and contained almost no albumin. This explains the contrast between the strong immunoreactivity of the neurons and the vague reactivity of the neuropil in light microscopy.Eight female Wistar rats (Merllegaard, Copenhagen, Denmark) weighing 200-250 g were used. The ani-

Section: Discussionsupporting

confidence: 92%

“…Six animals were killed in pairs after survival periods of 1 h, 6 h and 12 h, and blocks from the lesions were prepared for postembedding immunoelectron microscopy (9,19). These blocks were not postfixed in osmium tetroxide.…”

mentioning

confidence: 99%

Neuronal uptake of plasma proteins in cryogenic brain lesions

Løberg

Skjørten

et al. 1992

APMIS

“…This illustrates that IEM has higher sensitivity than IF in the sense that IEM is better suited for detecting small amounts of IgA deposits. This observation fits well with previous results on LR-white sections and highaccelerator epoxy sections [3,4].…”

Section: Diagnostic Powersupporting

confidence: 93%

“…However, IF microscopy has lower morphological resolution than ordinary light microscopy, and sometimes it is difficult to determine the position of the immune complex deposits within the glomeruli. Small immune complex deposits may also have low detectability by IF [3,4]. To overcome these problems, and to combine the ultrastructural preservation with immunolabeling of the immune complex deposits, some researchers have used an acrylic resin (LR-White) for embedding the renal biopsies for immunoelectron microscopy [4,5,6].…”

mentioning

confidence: 99%

A New Immunoelectron Microscopy Approach for the Detection of Immunoglobulin and Complement Deposits in E poxy-Embedded Renal Biopsies

Ultrastructural Pathology

Strøm

1998

Self Cite

The purpose of this study was to examine the diagnostic value of a new immunoelectron microscopy technique (IEM) for detection of immunoglobulin and complement deposits in epoxy-embedded renal biopsies. Twenty-four renal biopsies were embedded in epoxy resin following a tissue processing involving moderately increased amount of accelerator, DMP-30 (Tri(Dimethyl Amino Methyl) Phenol), in the infiltration steps. Following antigen retrieval by heating in citrate buffer, immunogold labeling was performed on ultrathin sections from these epoxy blocks with antibodies against immunoglobulins and complement. The sections were counterstained with urnayl acetate and lead citrate without any enhancing procedures. The preservation of the ultrastructure with this method was similar to that usually seen in epoxy embedded material. The immunogold labeling was intense and distinct. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. The correspondence between IF and IEM were good, but in some cases higher sensitivity for IgA with IEM than IF was observed in the sense that smaller amounts of antigen were detectable with IEM. The combination of moderately increased amount of accelerator and antigen retrieval is superior to previous methods with respect to ease of use, ultrastructural preservation, and intensity of the immunolabeling. Moreover, the renal tissue can be processed in an automatic ultraprocessor together with other specimens which are to be prepared for routine electron microscopy.

“…This illustrates that IEM has higher sensitivity than IF, in the sense that IEM is better suited for detecting small amounts of antigen. The observation that IEM is more sensitive for labeling IgA is substantial, since the presence of minor amounts of mesangial IgA may in some cases be the only finding establishing a diagnosis of IgA nephritis, and it fits well with previous results on LR-White sections (14). Indirect immunolabeling (IEM) may give more intense labeling than a direct procedure (IF).…”

Section: Iem As Diagnostic Toolsupporting

confidence: 86%

Immunoelectron microscopy on epoxy sections without deplasticizing to detect glomerular immunoglobulin and complement deposits in renal diseases

Strøm

Skjørten

1997

APMIS

Self Cite

Twenty renal biopsies were studied by immunoelectron microscopy (IEM) after embedding in epoxy resin. Immunogold labeling for immunoglobulins and complement C3 was performed on the epoxy sections, which were not subjected to any kind of etching or deplasticizing prior to the immunolabeling. The concentration of accelerator, DMP‐30 (Tri (Dimethyl Amino Methyl) Phenol), was increased in the infiltration and embedding steps far beyond the values normally used to make immunolabeling of these antigens possible on epoxy sections. The sections were stained with tannic acid accompanied by uranyl acetate and lead citrate. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. Some cases with IgA‐nephritis demonstrated a higher sensitivity for IEM than IF, in the sense that smaller amounts of antigen were detectable with IEM. Ultrastructural preservation with this method was approximately the same as that usually seen on epoxy‐embedded material. By combining excellent immunolabeling with nearly optimal ultrastructural morphology in one procedure, this method is useful particularly in situations where the material available is limited, such as in studies of renal biopsies. As far as we know, this is the first time that immunoglobulins have been satisfactorily immunolabeled on epoxy sections without etching or deplasticizing.