Degenerating Processes Identified by Electron Microscopic Immunocytochemical Methods

Abstract

The application of electron microscopic immunolabeling techniques to the identification and analysis of degenerating processes in neural tissue has greatly enhanced the ability of researchers to examine apoptosis and other degenerative disease mechanisms. This is particularly true for the early stages of such mechanisms. Traditionally, degenerating processes could only be identified at the ultrastructural level after significant cellular atrophy had occurred, when subcellular detail was obscured and synaptic relationships altered. Using immunocytochemical labeling procedures, degenerating neural and glial processes are first identified through the use of antibodies directed against a variety of degenerative markers, such as proapoptotic effectors (i.e., cytoplasmic cytochrome c), pathological components (i.e., beta amyloid deposits), or inflammatory agents (i.e., Iba1). Both the subcellular distribution of the marker within the process and the relationship of the labeled process to surrounding elements can then be carefully characterized. The information obtained can be further refined through the use of dual immunolabeling, which can provide additional data on the phenotype of the degenerating process and inputs to the process.

Hippocampal mossy fiber leu-enkephalin immunoreactivity in female rats is significantly altered following both acute and chronic stress

“…The portion of the forebrain containing the HF (from –2.0 to – 6.65 mm from Bregma) (Swanson, 1992) was then removed as a roughly 5-mm-thick coronal block that was further incubated in the latter fixative for an additional 30 min. Sections (40 μm thick) through the entire block were cut on a Vibratome (yielding on average 120 sections), collected in 0.1M PB, and transferred serially to tissue culture trays containing a solution (30% sucrose and 10% ethylene glycol in 0.1 M PB) for storage at –25° C. For a detailed description of the tissue preparation, immunocytochemical and analysis procedures used, refer to Milner et al (Milner et al, 2011). …”

“…Captured EM images (at 123x) of selected thin sections were then compared to captured LM images of the blockface before sectioning, allowing one to map the borders of the MF pathway within given thin sections. Since the extent of immunocytochemical labeling decreases with distance from the tissue surface, only grid squares within the MF pathway displaying no obvious tissue damage, but that were adjacent to the Embed812/tissue interface, were selected for analysis (Milner et al, 2011). For each animal/block, two thin sections were selected for analysis, and for each thin section, two to three such complete grid squares (3025 μm 2 per square) were selected at low magnification, on the basis of the positional criteria described, randomly, before visual inspection of the grid square field at a higher magnification.…”

Sex differences in NMDA GluN1 plasticity in rostral ventrolateral medulla neurons containing corticotropin-releasing factor type 1 receptor following slow-pressor angiotensin II hypertension

“…The blocks were then sectioned (40 μm thick) on a vibrating microtome (Vibratome; Leica) and collected in PB. Sections were stored in cryoprotectant (30% sucrose, 30% ethylene glycol in PB) at −20°C until immunocytochemical processing (Milner et al, 2011). …”

SorCS2-mediated NR2A trafficking regulates motor deficits in Huntington’s disease

“…Sections were processed for electron microscopy as previously described (91). Briefly, 12-month-old WT, zQ175 homozygous mice, or SorCS2 -/-mice were overdosed with sodium pentobarbital (150 mg/kg, i.p.)…”

“…SIG labeling for NR2A appeared as black electron-dense particles. The criteria for field selection and the measures to avoid false-negative labeling of smaller profiles, variability between animals in each experimental group, and differential reagent sensitivity comparing SIG and immunoperoxidase labeling were performed as described before (91,93). Profiles were considered dually labeled if they contained immunoperoxidase and at least 1 SIG particle.…”