Histochemical Use of Borohydrides as Aldehyde Blocking Reagents

Complete blocking of the Schiff reaction applied after HIO₄ oxidation is attained by 1%, 0.5% or 0.2% NaBH₄ in 1% Na₂HPO₄ with a 1 min exposure, 0.1% NaBH₄ requires 2 min. KBH₄ was also completely effective in the same solvent at 1, 0.5 and 0.25% in 1 min. The solutions deteriorate on standing, so that 0.5% NaBH₄ is effective in 1 min at 7 hr but 5 min is needed at 24 hr and is ineffective at 36 hr; 1% KBH₄ requires 4 min at 24 hr when fresh, and showed deterioration in 2-4 days. Saturated (0.4%) isopropanol and 1% pyridine solutions required 10-15 min when fresh and showed deterioration in 2-4 days. No satisfactory results were obtained with 80% or absolute dioxan or with methyl cellosolve in times of an hour or less; even a 24 hr exposure was ineffective with 0.2% in 80% dioxan.     

Histochemical Demonstration of Pyrophosphatase

Fresh tissue slices were fixed in 5% formalin containing 0.9% NaCl for 10-20 min and frozen sections therefrom floated for 3 hr at 37°C on an incubating mixture made as follows. Sodium pyrophosphate (Na₄P₂O₇-12H₂O), 1.088 gm was dissolved in 20-30 ml of distilled water and to this was added ferric chloride (FeCl₃-6H₂O), 0.61 gm dissolved in 10-15 ml of water. The precipitate was just dissolved by cautiously adding 5-10% aqueous Na₂CO₃ solution and the pH adjusted to 7.2 with 1N HCl. The volume was made up to 100 ml and 0.9 gm of NaCl added. Before use, 1 ml of 10% Mg(NO₃) was added. After incubation, sections were washed 10-15 min in 0.9% NaCl, then mounted on glass slides and air-dried. When dry, the slides were immersed in 0.9% NaCl containing 0.2-0.5% ammonium sulfide for 2-3 min, then dehydrated rapidly through graded alcohols, cleared, and covered in balsam. Sites of pyrophosphatase activity stained in various shades of green. Acid pyrophosphatase also was histochemically demonstrated by the same principle, excepting that the substrate solution was adjusted to pH 3.7-4.0 with acetate buffer. The pattern of distribution of pyrophosphatase and glycerophosphatase was almost identical.     

Histochemical Study of Aldehyde Dehydrogenase in the Rat CNS

A quantitative histochemical method was developed to determine aldehyde dehydrogenase (EC 1.2.1.3; ALDH) activity in the CNS. The distribution of ALDH activity in all rat brain and spinal cord regions is described. Among the CNS neuron structures, high enzyme activity was found in receptor and effector neurons, whereas low activity was noted in perikarya of the majority of intermediate neurons, including all aminergic neurons. A positive correlation was demonstrated between the distribution of ALDH activity among rat CNS microregions (our own data) and the density of dopaminergic terminals, dopamine content, and monoamine oxidase activity (literature data) among the same microregions. They may reflect a spatial linkage between ALDH and the predicted sites of natural aldehyde production. Lower enzyme activity was found in phylogenetically younger brain structures. It may explain the differential resistance of CNS structures to ethanol (acetaldehyde). Among the barrier CNS structures, moderate ALDH activity was found in capillaries and surrounding astrocytes and high activity was noted in ependimocytes covering the brain cavities and those of the vascular plexus. This provides realization of the function of ALDH as a brain metabolic barrier for aldehydes.     

Simplified Manufacture and Histochemical Use of the Schiff Reagent

Schiff reagents were made by two methods. The first procedure gave a Schiff reagent of pH 1.8-2.4. It was accomplished by passing sulfur dioxide into 0.5% aqueous fuchsin solution at room temperature, stopping at reddish violet, and decolorizing allowed to occur on standing. In another method, 1.5 ml. of 5.6% sulfurous acid was added to 100 ml. 0.5% fuchsin solution and the mixture produced in several hours a colorless Schiff reagent of pH 3. The solution remained unchanged for some weeks when kept stoppered in a refrigerator.

To test these Schiff reagents, histochemical examinations were carried out with Feulgen and McManus reaction in various pH ranges. These experiments showed that the Feulgen reaction was optimum at pH 3, the McManus reaction at pH 2.4.     

Diacetyl for Blocking the Histochemical Reaction for Arginine

Two different histochemical methods for blocking the guanidinium group of arginine in formalin fixed, paraffin embedded material are reported here. One procedure uses diacetyl with short incubation times and high pH, the other uses the trimer of the diacetyl reagent and requires longer incubation times but moderate pH. The diacetyl reagent is recommended despite its high pH because the preparation of the diacetyl trimer is laborious and time-consuming.     

Histochemical Demonstration of Carbonic Anhydrase Activity

Fresh tissue slices fixed in chilled acetone for 1 hour and washed in distilled water for 10-30 minutes were incubated for 30-45 minutes at 37°C. in the freshly prepared incubating mixture: filtrate of a mixture of 8% sodium bicarbonate, 100 ml., and MnCl₂·4H₂O, 1 g. After washing in distilled water for 1 hour, they were dehydrated and embedded in paraffin. Sections were cut 15-20μU, deparaffinized, rinsed in absolute alcohol and placed in a 0.1% solution of potassium periodate for 48 hours at 37°C. The mounted sections were counterstained (if desired), dehydrated in alcohol, cleared in xylene (not carbol-xylene) and mounted in balsam. Many brown granules were produced on the sites of enzyme activity by this procedure. The results obtained seem to be in good agreement with previous findings by biochemical determinations.     

Histochemical Demonstration of Protein-Bound Alpha-Acylamido Carboxyl Groups

A method has been developed to demonstrate the alpha-acylamido carboxyl groups of protein, taking advantage of the fact that acylamido carboxyl groups are converted to ketonic carbonyls by the action of acetic anhydride and absolute pyridine. The method utilizes deparaffinized sections of tissues fixed in a variety of fixatives. Following the conversion of carboxyls to the methyl ketones, the latter are stained with 2-hydroxy-3-naphthoic acid hydrazide. Control experiments have indicated that methylation of carboxyls prevented staining, as did carbonyl reagents after the carboxyls were transformed to methyl ketones. Leucofuchsin did not stain the ketonic carbonyls, and only elastic tissue stained with 2-hydroxy-3-naphthoic acid hydrazide without the previous use of the catalyzed reaction with anhydride. A brief survey of the reaction on various tissues of the albino rat was made, and the effects of various fixatives were assayed. Of particular interest were certain sites, such as acidophiles of the anterior pituitary gland, where an intense reaction occurred. The possibility exists that certain specific proteins rich in terminal acylamido carboxyl groups, by virtue of their protein side chains or low molecular weight, may be demonstrated by this method.     

Histochemical Demonstration of Acid Phosphatase in Hard Tissues

The action of the following decalcifying solutions for the demonstration of acid phosphatase has been studied: buffer solution acetic-acetate 0.05 M, pH 5; 2, 5, 10, 20, and 50% formic acid and 20% sodium citrate in equal parts (pH 2.6, 3, 3.8, 4.2, and 5); 0.5 M citric acid-NaOH, pH 4.2; Versene solution, 5%, pH 7. A comparative study of fixatives has been made also (neutral formalin, 10-20%; formalin-chloral hydrate (Fishman), acetone and 80% alcohol). The best results were obtained with fixation at 4°C in 10-20% neutral formalin or formalin-chloral hydrate, for a period of 24 hr, and decalcification with 20% sodium citrate, 5% formic acid, in equal parts, pH 4.2, which can act on both specimens or sections for a period up to 2 wk with very little loss of enzyme. It is not necessary to reactivate the enzyme after decalcification; frozen sections should be used and should be washed in distilled water before proceeding with the demonstration of the enzyme (Gomori's method or azo dye coupling). Other fixatives (acetone and alcohol) and paraffin embedding produce a greater loss in enzymes and very irregular results.     

Factors Influencing the Histochemical Demonstration of Coenzyme-Dependent Dehydrogenases and Diaphorases

Procedures for the histochemical demonstration of DPN and TPN diaphorases have been presented by other workers. These techniques rely on the coenzyme-dependent dehydrogenases present in the tissue slice to generate the substrate required by the diaphorases. In vitro studies were carried out on kidney and adrenal tissue of the rat, using NT (neotetrazolium) and INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) with various substrates of DPN-dependent dehydrogenases. The solutions used for study contained alcohol and alcohol dehydrogenase, glutamate and malate, malate, glutamate, β-hydroxybutyrate, or DPNH. It has been possible to demonstrate (1) that histological distribution of dehydrogenases may differ from that of the flavoprotein oxidizing reduced coenzyme I; (2) characteristic patterns of distribution of particular dehydrogenases in the tissue proper; (3) different levels of dehydrogenase in kidney and adrenal; and (4) differences in dehydrogenase distribution in the kidneys of man and rat. The evidence presented clearly indicates the limitations inherent in the accepted procedures for the demonstration of DPN and TPN diaphorases. The possible application of the tetrazolium salts to the study of particular coenzyme-dependent dehydrogenases and the pitfalls which might occur are also discussed.     

Histochemical Demonstration of type IV collagen in the renal glomerulus

Summary Disulfide-groups are demonstrated in the basement membranes of glomerular capillaries, proximal convoluted tubules, and collecting ducts by means of a thiosulfation/Alcian Blue +0.8 Mol MgCl₂-staining sequence. It is suggested that the reaction shows type IV collagen of basal lamina material, which is characterized by a relatively high cystine content (8 half-cystine residues/1000).     

A Histochemical Method for the Demonstration of Diphosphopyridine Nucleotide Diaphorase

The present investigation concerning the histochemical demonstration of DPN diaphorase follows the development of a new reagent, Nitro-BT, which has already been used successfully for the cytochemical localization of the succinic dehydrogenase system. The most consistently favorable results were obtained with the lactate-lactic dehydrogenase system buffered at pH 7.4. Using sections of rat kidney and stomach, it was found that the intensity of stain was optimal after 15 minutes incubation at 37°C., conducted aerobically. By appropriate variations in the substrate mixture it was possible to selectively demonstrate the histochemical distribution of certain DPN-linked dehydrogenases in addition to DPN diaphorase. This was made possible by the special distribution of some of these dehydrogenases which distinguished them from one another. Of the dehydrogenases studied the distribution pattern of β-hydroxybutyric dehydrogenase was the most singular. In the gastric mucosa β-hydroxybutyric dehydrogenase was restricted to the cells of the mucous lining epithelium and the gland necks; and in the kidney the enzyme was limited to the cells of the proximal convoluted tubule and thick limbs of Henle's loop. In contrast, lactic dehydrogenase like DPN diaphorase was demonstrable in almost all cytologic elements of both the stomach and the kidney.     

Histochemical Demonstration of Endogenous Dehydrogenase Systems by Supravital Perfusion

Endogenous dehydrogenase activity is demonstrated in fresh, intact organs by supravital perfusion with a tetrazolium solution. The animal is first injected intravenously with 1.5 mg Heparin/100 gm body weight. It is then anesthetized and a fine polyethylene cannula (PE50, Intramedic) is inserted into a major artery and secured with a ligature. An initial perfusion with warm (37°C) M/20 phosphate buffer (pH 7.6) to remove the blood from the tissues is followed by a 10 min perfusion with the same kind of buffer to which has been added 0.25% neotetrazolium chloride (Dajac Laboratories). The tetrazolium solution is delivered to the tissue at the rate of 1 ml/minute. A final perfusion with 10% formalin in warm phosphate buffer (pH 7.6) flushes and fixes the tissues. Frozen sections can then be cut and mounted in glycerol jelly. Fine, colored formazan crystals are deposited at the sites of enzyme activity. The method is simple and yields excellent histochemical preparations.     

Histochemical Demonstration of Succinic Dehydrogenase in Ascites Tumor Cells

Fresh undiluted tumor ascites (0.05 ml) withdrawn from peritoneal cavity was placed immediately in a centrifuge tube containing 2.0 ml of an aqueous mixture prepared with 1 part each of the following solutions: 1% neotetrazolium chloride, 0.2 M sodium succinate and 0.1 M phosphate buffer, pH 7.4. The tube was incubated for 2 hr at 37°C and centrifuged for 3 min at 700 rev/min. The precipitate was washed with 0.85% saline solution and subsequently fixed with neutral 10% formalin for 10 min. After centrifugation, smears or squash preparations of the precipitate were prepared. Succinic dehydrogenase activity was demonstrated very distinctly and uniformly by the granular deposition of a deep purple pigment intracellularly.     

Blocking integrin inactivation as an anti‐angiogenic therapy

During angiogenesis, endothelial cell migration is coordinated by integrin‐mediated contact with the extra‐cellular matrix (ECM), coupled with receptor tyrosine kinase signalling to regulate dynamic cytoskeletal and plasma membrane reorganization. A recent paper by Vitorino et al ( 2015 ) defined a new MAP4K4–moesin–talin–β1‐integrin pathway that could be therapeutically exploited to suppress pathologic angiogenesis.     

Agro-Successional Restoration as a Strategy to Facilitate Tropical Forest Recovery

With the increasing need to restore former agricultural lands worldwide and in the tropics, in particular, it is critical to explore different models for how to restore these lands in a cost-effective manner which best simulates natural forest recovery and provides for human livelihoods. We propose that agro-successional restoration, which we define as incorporating a range of agroecology and agroforestry techniques as a transition phase early in forest restoration, could be used more widely to overcome socioeconomic and ecological obstacles to restoring these lands. Over centuries, farmers and scientists have developed various agroforestry techniques that aim to cultivate crops and trees, in a range of crop types, time periods of cultivation (a few years to several decades), and complexity of species planted. The management practices used in these systems, such as weeding and increasing soil fertility, parallel those used in many forest restoration efforts. The synergism between these approaches is evidenced by many existing agro-successional examples currently used by smallholders in the tropics. Benefits of the agro-successional model include extending the management period of restoration, offsetting some management costs, providing food security for small landholders, and involving small landholders in the restoration process.     

Calibration of Quantitative Histochemical Methods: Estimation of Glycogen Content of Muscle Fibers Using the PAS Reaction

A fairly simple method for calibrating microdensitometric histochemical assays is described. The method is based on paired biochemical and histochemical assays on single freeze-dried skeletal muscle fibers which differ widely in their properties. As an example, the method is applied to investigate the validity of the periodic acid-Schiff (PAS) reaction for the microdensitometric estimation of glycogen content. Some problems that may interfere with the calibration are discussed.     

The Histochemical Demonstration of Hemoglobin in Blood Cells and Tissue Smears

The demonstration of intracellular hemoglobin in permanent preparations has long been a problem. The affinity of hemoglobin for iron hematoxylin is well known but this stain also colors yolk, chromatin, and other structures and is therefore not a reliable criterion. The presence of hemoglobin has been associated with an acidophil cytoplasm which stains a characteristic color, but a careful inspection of living cells in early hematopoetic or embryological stages demonstrates that hemoglobin is present in the erythrocytes which are quite basophilic. In the course of some research on the blood of embryonic frogs it became desirable to demonstrate the presence of hemoglobin in cells by means of a specific staining reaction.     

Optimal Parameters for the Histochemical Demonstration of Acetylcholinesterase in Plastic Sections of Rat Brain

A modified method for improved preservation and optical resolution of acetylcholinesterase (AChE)-containing structures in adult rat brain is described. Optimal tissue preparation included fixation in paraformaldehyde 4%, glutaraldehyde 0.1%, and sucrose 7% in 0.1 M Sorensen's phosphate buffer, pH 7.4, rinsing in buffer 50 mM with respect to NLL, Q and 2% with respect to sucrose, acetone dehydration, vacuum infiltration widi LKB Historesin, and polymerization at 4 C, overnight incubation of 10 μm sections at 37 C in the AChE histochemical reaction mixture and silver intensification according to Hedreen et al. Demonstration of AChE enzyme activity in the cholinergic projection from the rat basal forebrain to the ipsilateral hippocampus exemplifies the usefulness of the technique. The method provides an excellent demonstration of AChE-positive axonal processes and enables the pharmacohis-tochemical visualization of cholinergic neurons. This procedure offers a convenient method for analysis of cholinergic neurons that avoids potential artifacts inherent in other AChE histochemical procedures.