A Simplified Method for the Histochemical Detection of Iron in Paraffin Sections: Intracellular Iron Deposits in Central Nervous System Tissue

Although all cells contain iron, most histochemical methods fail to reveal the presence of iron within many cells of the central nervous system (CNS), particularly neurons. Previously, a sensitive method was developed that limited the extraction of iron in paraffin sections, and this method revealed staining within neurons. However, the staining was often too robust making it difficult to discern discrete intracellular structures. In 1970, a study incorporated acetone in an iron histochemical procedure to facilitate the demarcation of staining features. In the present study, both acetone and limits to iron extraction were included in a simplified staining procedure. This procedure was applied to paraffin sections of CNS tissue from CISD2 deficient and littermate control mice. Discrete nuclear and cytoplasmic staining features were detected in all mice. Although widely present in neurons, punctate cytoplasmic staining was particularly prominent in large neurons within the hindbrain. Evaluation of extended depth of focus images, from serial focal planes, revealed numerous stained cytoplasmic structures. Additionally, the simplified staining procedure was applied to paraffin sections from Alzheimer’s disease and control cases. Despite suboptimal processing conditions compared to mouse tissue, discrete staining of cytoplasmic structures was revealed in some neurons, although many other neurons had nondescript staining features. In addition, initial findings revealed iron deposited within some vessels from patients with Alzheimer’s disease. In summary, since paraffin sections are commonly used for histological preparations, this simplified histochemical procedure could facilitate the study of iron in various CNS conditions by revealing staining details often missed by other procedures.

Reserve capacity for ATP consumption during isometric contraction in human skeletal muscle fibers

Maximum velocity of the actomyosin ATPase reaction ( V max ATPase) and ATP consumption rate during maximum isometric activation (ATPiso) were determined in human vastus lateralis (VL) muscle fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that the reserve capacity for ATP consumption [1 − (ratio of ATPiso to V maxATPase)] varies across VL muscle fibers expressing different MHC isoforms. Biopsies were obtained from 12 subjects (10 men and 2 women; age 21–66 yr). A quantitative histochemical procedure was used to measure V max ATPase. In permeabilized fibers, ATPiso was measured using an NADH-linked fluorometric procedure. The reserve capacity for ATP consumption was lower for fibers coexpressing MHC2X and MHC2A compared with fibers singularly expressing MHC2A and MHCslow (39 vs. 52 and 56%, respectively). Tension cost (ratio of ATPiso to generated force) also varied with fiber type, being highest in fibers coexpressing MHC2X and MHC2A. We conclude that fiber-type differences in the reserve capacity for ATP consumption and tension cost reflect functional differences such as susceptibility to fatigue.

High-resolution localization of acetylcholinesterase at the rat neuromuscular junction.

To overcome the limited ultrastructural resolution of conventional acetylcholinesterase (AChE) ultrahistochemistry, acetylcholine (ATCh) was used to reduce the rate of enzymic thiocholine liberation. The conventionally limited resolution is mainly due to the high focal activity of the enzyme in neural structures, because cleavage of substrate is faster than histochemical trapping reactions. Therefore, using the copper-thiocholine method, we investigated the reduction of thiocholine liberation by acetylcholine (ACh). As examined biochemically, the apparent Ki for ACh was close to the Km for ATCh. The ACh/ATCh ratio, therefore, determined the reduction of thiocholine production in histochemical experiments. In addition, the morphological appearance of the precipitated reaction product after its changes during the histochemical procedure was monitored using electric eel AChE immobilized on Sepharose 4B. The improved fine structural resolution at 40- to 100-fold excess of ACh over ATCh is demonstrated at the neuromuscular junction of rat lumbricalis muscle. The highest focal enzyme activity is found at the presynaptic membrane and in the secondary cleft, but not on top of the junctional folds, indicating the separation of esterase and nicotinic receptors. The physiological events during neuromuscular transmission are discussed on the basis of the new "gradient switch hypothesis" suggested in this report.

Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure.

A sensitive method for acetylcholinesterase (AChE) histochemistry has been developed which permits simultaneous observation of fine fiber processes and neuron cell bodies. In rat brain, distinctive configurations can be observed which have been difficult to see by other techniques. The staining procedure involves two steps. Tissue sections are incubated first in Karnovsky and Roots medium diluted one-hundredfold; and then with a mixture containing diaminobenzidine (DAB) and H2O2. The reaction product of the first step induces cleavage of hydrogen peroxide in the second step, with a resulting oxidation of DAB to yield a fine precipitate. Addition of metal ions, such as nickel, to the DAB-H2O2 mixture produces high-contrast, Golgi-like images of neuron structures. The technique is much more sensitive than previous methods and greatly reduces background staining caused by crystallization of reaction products. Many potential applications exist for this new technique, in addition to the initial results described here.

Immunocytochemical and enzyme histochemical localization of kallikrein-like enzymes in colon, intestine, and stomach of rat and cat.

Kallikrein was localized in goblet (or mucous) cells of rat colon and in rat and cat small intestine and stomach by two immunocytochemical techniques. A kallikrein-like enzyme was also localized by enzyme histochemistry in mast cells of colon, intestine, and stomach of the cat, where they appeared to be associated with blood vessels in the lamina propria. The mast cell enzyme, however, was not detected by immunocytochemistry using antibodies to kallikrein. Modification in the enzyme histochemical procedure (pH, fixation) yielded positive results for a kallikrein-like protease in goblet cells of the intestine and colon. The possible physiological and pathological significance of kallikrein-like enzyme in the gastrointestinal tract and elsewhere is discussed.

Biochemical and cytochemical evidence indicates that coated vesicles in chick embryo myotubes contain newly synthesized acetylcholinesterase.

We have isolated highly purified coated vesicles from 17-d-old chick embryo skeletal muscle. These isolated coated vesicles contain acetylcholinesterase (AChE) in a latent, membrane-protected form as demonstrated enzymatically and morphologically using the Karnovsky and Roots histochemical procedure (J. Histochem. Cytochem., 1964, 12:219-221). By the use of appropriate inhibitors the cholinesterase activity can be shown to be specific for acetylcholine. It also can be concluded that most of the AChE represents soluble enzyme since it is rendered soluble by repeated freeze-thaw cycles. To determine the origin of the coated vesicle-associated AChE, we have isolated coated vesicles from cultured chick embryo myotubes which have been treated with diisopropylfluorophosphate, an essentially irreversible inhibitor of both intra- and extracellular AChE, and have been allowed to recover for 3 h. This time is not enough to allow any newly synthesized AChE to be secreted. These coated vesicles also contain predominantly soluble AChE. These data are compatible with the hypothesis that coated vesicles are important intermediates in the intracellular transport of newly synthesized AChE.

Ultrastructural and cytochemical demonstration of peroxisomes in cultured fibroblasts from patients with peroxisomal deficiency disorders.

The oxidation of very long chain fatty acids and synthesis of ether glycerolipids (plasmalogens) occurs mainly in peroxisomes. Zellweger's cerebrohepatorenal syndrome (CHRS) is a rare, inherited metabolic disease characterized by an apparent absence of peroxisomes, an accumulation of very long chain fatty acids, and a decrease of plasmalogens in tissues and cultured fibroblasts from these patients. As peroxisomes are ubiquitous in mammalian cells, we examined normal and CHRS-cultured fibroblasts for their presence, using an electron microscopic histochemical procedure for the subcellular localization of catalase, a peroxisomal marker enzyme. Small (0.08-0.20 micron) round or slightly oval peroxisomes were seen in both normal and CHRS fibroblasts. The number of peroxisomes was analyzed morphometrically and found to be significantly reduced in all CHRS cell lines. These results are discussed in relation to the underlying defect in peroxisomal function and biogenesis in this disease.

Ontogeny of Cells Containing Insulin, Glucagon, Pancreatic Polypeptide Hormone and Somatostatin in the Bovine Pancreas

Antibodies to insulin, glucagon, pancreatic polypeptide hormone (pPj and somatostatin were used in the immunofluorescence histochemical procedure to study the ontogeny of pancreatic endocrine cells containing the four hormones in the bovine fetus of approximately 100 days gestation to term. Pancreatic sections from the bovine neonate and adult were also examined for the cellular distribution of the four hormones. Immunoreactive cells staining for insulin, glucagon, PP and somatostatin were present in the pancreas of all fetuses studied. Each endocrine cell type displayed a characteristic distribution within the developing pancreas and in the neonate and adult. The presence of the four islet hormones relatively early in bovine fetal life suggests that they may be important in intra- and extra-islet metabolism in the fetus.