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

Abstract

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.

A rapid method for generating cystic forms of Borrelia burgdorferi, and their reversal to mobile spirochetes

“…TEM was performed using the following procedure: The culture was centrifuged at 14,OOOXg for 20 min. The medium was removed and replaced with 2% glutaraldehyde in 0.2 M cacodylate buffer (pH= 7.3); the bacteria were fixed in this fixative for 2 h and postfixed in 1% osmium tetroxide in 0.2 M cacodylate buffer for 2 h. The pellet was dehydrated, infiltrated, and embedded in conventional epoxy resin (LX-112 ; Ladd, Burlington, VT, USA) using a previously described method (38,39). Ultrathin sections were cut with a diamond knife (Jumdi; Juniper ultra Micro, Stockholm, Sweden) on an ultramicrotome (LKB 2088 Ultrotom V) and mounted on 200 mesh copper grids.…”

‘Airborne’ contact dermatitis due to Leica immersion oil

“…Methacrylate embedding followed by resin removal facilitates visualization of the Golgi apparatus or the cytoskeleton 19 , 20 . In nephrology, renal biopsies are studied by immunoelectron microscopy after embedding in epoxy resin 21 …”

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine

“…The examination by TEM was carried out according to the following procedure. The cultures were centrifuged (5,000 g, 20 min), the medium was removed and replaced with 2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3), and the bacteria were fixed for 2 h. The bacteria were post-fixed in 1% osmium tetroxide in 0.2 M cacodylate buffer for 2 h. The pellets were dehydrated, infiltrated and embedded in conventional epoxy resin (LX-112; Ladd, Burlington, Vt., USA) by a method described earlier [9,10]. Ultrathin sections were cut with a diamond knife (Jumdi; Juniper ultra Micro, Stockholm, Sweden) on an ultramicrotome (LKB 2088 Ultrotom V) and mounted on 200-mesh copper grids.…”