Journal of Histochemistry & Cytochemistry

Using histochemical techniques it has been demonstrated that the adrenal glands of baby rabbits lack noradrenaline and produce only adrenaline, while the adrenal glands of the pigeon contain oth adrenaline and noradrenaline and the relative proportion of noradrenaline secreting cells is almost the same as that of the adrenaline secreting cells. On exposure to higher ambient temperature there is release of adrenaline from the adrenal glands of baby rabbits and of both adrenalin and noradrenaline from the adrenal glands of pigeons. The possible role of the hypothalamus and the sympathetic nervous system in this response is discussed.

Nuclear localization of succinic dehydrogenase is being reported in normal and malignant cells from epidermoid carcinomatous tissue of human cervix. This is a new finding apart from its well established cytoplasmic counterpart.

A procedure was described for demonstrating acidic polysaccharides in formalin-fixed paraffin-embedded tissue sections using fluorescein-labelled Aspergillus polysaccharide. This material is a basic polysaccharide composed of units of galactosamine and N-acetyl galactosamine and is synthesized by the mold Aspergillus parasiticus.

The distribution of acidic polysaccharides shown by the fluorescein-labelled Aspergillus polysaccharide method was similar to results with the alcian blue-periodic acid Schiff sequence and to those with fluorescein labelled deacetylated chitin. Both epithelial and connective tissue mucins were demonstrated by the fluorescein-labelled basic polysaccharide.

Aspergillus polysaccharide may be considered a colorless polyvalent cation that acts as a carrier for the fluorochrome in this procedure. Since it can react with many types of biological cells as well as with various substances isolated from cells, it may have other important applications in histochemistry and cytochemistry.

A method for demonstrating acidic polysaccharides in formalin fixed parafin embedded tissue sections using fluorescein labelled deacetylated chitin is described. Epithelial and connective tissue acidic polysaccharides have been studied in a number of organs. The distribution of acidic polysaccharides as seen with fluorescein-labelled deacetylated chitin is similar to results obtained with alcian blue-periodic acid Schiff and fluorescein-labelled Aspergillus polysaccharide. Previously reported fluorescent staining methods for acidic polysaccharides such as iron-hematoxylin-acridine orange and atabrine as well as fluorescein-labelled deacetylated chitin may prove to be useful adjuncts to the light microscopic methods of demonstrating mucins. Other possible uses of deacetylated chitin, a colorless macro-cation, in histochemistry and cytochemistry are noted.

The effect of basic polysaccharides on the staining characteristics of acidic polysaccharides in tissue sections of formalin-fixed paraffinembedded material has been studied. Acidic polysaccharides ordinarily do not stain with acidic dyes, but stain well with basic dyes. The complex formed when a basic polysaccharide such as Aspergillus polysaccharide or deacetylated chitin reacts with acidic polysaccharides assumes the staining characteristics of the basic polysaccharide, that is, it stains with acidic dyes. Thus, areas in tissue sections containing acidic polysaccharides can be colored with acidic dyes. Further, the basic polysaccharides were shown to temporarily block staining of acidic polysaccharides by alcian blue and mucicarmine.

Solutions of the basic compounds, Aspergillus polysaccharide and deacetylated chitin were tested for their ability to precipitate various materials synthesized by biological cells. All acidic (anionic) polysaccharides tested were precipitated, but non-polar polysaccharides like glycogen and dextran failed to react. Both basic polysaccharides were also able to precipitate ribonucleic acid and deoxyribonucleic acid as well as several proteins.

Both polysaccharides agglutinated a variety of bacteria and yeasts as well as erythrocytes and liver, kidney, spleen, sperm and tumor cells of mammalian origin. The significance of these observations was discussed.

The author has studied the occurrence of periodic-acid-Schiff-positive materials in the vaginal and cervical epithelia of albino rats. The Müllerian ducts first lack periodic-acid Schiff-positive substances; later there occurs a storing of glycogen in the Müllerian epithelium In immature animals the glycogen is gradually replaced by saliva resistant periodic-acid-Schiff-positive materials. It adult animals glycogen has been found in the stratified cervical epithelium only in proestrus and early estrus.

A method for demonstrating nuclear ribonucleic acid is given. This entails the use of Grams' stain of heat fixed tissues. The selectivity of crystal violet for nuclear ribonuclei acid suggests a fundamental difference between the nuclear and cytoplasmic varieties of this substance.

Photometric restudy of 120 Feulgen-stained bovine spermatozoa after an interim period of 30-38 days from their initial date of measurement clearly demonstrates a marked decolorization of the cells, depending on the particular lot of Feulgen reagent used. With an eight-month-old and often-used reagent, no fading occurred over a period of 38 days. Using a fresh reagent, made with the same batch and initial content of basic fuchsin as the above, a fading of 14.6% was observed in 30 days, and of 20.9% in 37 days. These findings clearly emphasize the need for greater control of the Feulgen reagent and reaction and for the caution that must be exercised in using long prepared Feulgen-stained material for quantitative purposes.

The application of a number of enzyme and other histochemical tests, and of cytological techniques to rat spinal ganglion cells has demonstrated a wide range in intensity and distribution of these reactions in individual cells. Since all stages between one type of intensity and distribution and another can be seen it is suggested that the spinal ganglion cells undergo continuous metabolic cycles which are related possibly to the synthesis of protein, acetyl choline, cholinesterase and perhaps other enzymes and compounds.

A comparative study was made of the distribution of mitochondrial adenosine triphosphatase activity in several organs of rat and rabbit using both the calcium and lead techniques. Certain modifications in these techniques assured both improved intracellular morphology and considerable preservation of enzyme activity. Of particular importance was the low temperature (–2 to –3°C) of the neutral calcium-formol solution, and, following short fixation (lead method), a subsequent wash in neutral buffer. Mitochondrial activity was similar with both techniques, but the lead method proved to be less costly, less time-consuming, and above all, far less capricious than the calcium technique. In good preparations, the appearance of mitochondria is as clearly defined as in sections stained with non-enzymatic, conventional techniques. Long exposure of cryostat sections to formalin or preparation of sections from tissue blocks fixed in neutral formalin leads to the complete abolition of mitochondrial activity; on the other hand, it accentuates enzyme staining of other structures, such as, for instance, bile canaliculi in the liver and the secretory capillaries in the pancreas and salivary glands. It also visualizes the infolding membranes in certain tubules of the rat and dog kidney. It is assumed that formalin-fixation aids in the enzymato-morphologic distinction of these two different intracellular structures.

Manometric studies have revealed that the oxidation of p-aminodiphenylamine by Keilin and Hartree beef heart muscle particles was several-fold higher in the presence of added cytochrome c than in the absence of the pigment. The oxidation of p-aminodiphenylamine was inhibited 90% by 10^–4 M cyanide. Correlated spectrophotometric studies demonstrated that the oxidation of p-aminodiphenylamine by Keilin and Hartree particles and fresh-frozen dehydrated tissue sections produced substances with identical visible and ultraviolet absorption spectra. The above experiments further indicate that the oxidation of p-aminodiphenylamine is mediated via cytochrome oxidase, and that this reagent provides valid histochemical localizations of the respiratory enzyme.

The influence of formaldehyde on rat brain lipid was investigated by comparing the analysis of lipids from fresh rat brain with the analysis from rat brains fixed for 110 days in a formaldehyde solution.

A quantitative analysis of the lipids using column and paper chromatography is described.

Sphingomyclin, cerebrosides and sulphatides were not attacked. Only a slight diminution of phosphatidylserine was indicated. Phosphoinositides remain fairly constant. Of lecithin about 25% was converted to lyso-lecithin. The phosphatidylethanolamine and lyso-phosphatidylethanolamine fractions suffered a marked diminution and the reaction with formaldehyde was noticeable in a change of chromatographic behaviour. The fraction containing cholesterol, cholesterol esters, glycerides, and fatty acids increased, the increase being correlated with the decrease of other fractions by the formation of lyso compounds.

After storing tissues for months in formaldehyde solutions a reliable analysis of the lipid content is still possible.

A new method for the histochemical demonstration of unsaturated lipids having a sounder chemical basis than previously reported methods has been developed. Formalin fixed frozen sections are treated with aqueous KBr₃; silver bromide is precipitated at the site of bromination by treatment with silver nitrate in dilute nitric acid; free silver is then formed by reduction with a photographic developer. Specificity is shown by the abolition of staining following any of these control reactions: a) elimination of the bromination step; b) prior blockade of double bonds with performic acid; c) extraction of lipids with chloroform-methanol or d) extraction of silver bromide before development with thiosulfate or cyanide solutions. In addition to normal tissues, atherosclerotic tissues and sections of central nervous tissues exhibiting lipid dystrophies have been examined. This technique has been fully demonstrated on pure compounds deposited on filter paper. Hydrogenated or performic acid treated compounds give negative results.

To obtain additional insight into the chemical reactions occuring furing fixation of tissue lipids by osmium tetroxide, the reaction between osmium tetroxide and lecithin has been studied. Procedures have been developed whereby quantitative information may be obtained about the nature of this reaction.

One series of experiments was designed to arrive at the stoichiometry of the reaction and its time-course. To this end air-dried lecithin spots on cover glasses or on filter paper were exposed to oxmium tetroxide vapor. The weight increase of the lecithin spots was determined, and related to time of exposure to osmium tetroxide and to the quantity of lecithin present. Under the conditions chosen the binding of osmium tetroxide rapidly reaches a limiting value; thus the reaction appears to proceed to a definite end-point. The number of double bonds in the lecithin molecule, as determined by gas chromatography, corresponds with the number of moles of osmium tetroxide bound per mole of lecithin. This supplements earlier spectrophotometric findings, and confirms part of the reaction scheme proposed by Criegee for the interaction between osmium tetroxide and unsaturated compounds generally.

The chemical properties of lecithin, and of other polar lipids, are determined not only by the presence of unsaturated linkages but also by the presence of an ampho-ionic group. Earlier research has demonstrated that this group is responsible for the staining properties of lecithin with anionic dyes, for instance, Brilliant Scarlet 3 R (Ponceau 4R or 4RS, Colour Index No. 16255). In this paper evidence is presented to show that the stainability of lecithin spots by Brilliant Searlet 3 R, under specified conditions, decreases markedly with increasing time of exposure to osmium tetroxide. It appears likely that as a result of the reaction with osmium tetroxide the lecithin polar group has changed properties.

After studying the staining properties of lecithin towards an anionic dye, in relation to time of exposure to osmium tetroxide, its staining capacity for a representative cationic dye was similarly studied. Evidence is presented demonstrating an increased binding capacity of exposed lecithin spots to Brilliant Green (Colour Index no 42040). After considering a few possible interpretations we suggest that the simplest hypothesis to account for this phenomenon is a partial removal of the positive charge of lecithin, whereby the negatively charged groups are freed for interaction with positive dye molecules.

Some sources of significant error in quantitative cytochemical work are discussed and methods for their control and elimination described. These recommendations are based on a study of distortion and thickness variation in histological sections of embedded biological tissues. To achieve the objectives of the investigation, it proved necessary to manufacture special microtome knives having standardized properties and to develop an optico-mechanical procedure for topographic mapping of section thickness. Altogether 10,500 thickness determinations were made on 140 sections, there being 10 similar sections in each of 14 series which differed with respect to tissue type, embedding substance, microtome drive and feed setting. The results disclosed that, even under ideal sectioning conditions and irrespective of tissue type or embedding substance, considerable variations in thickness must be anticipated, particularly between sections in a series. The mean coefficient of variation for the deviations between and within sections nominally 5 µ thick was 28% and 5% respectively.

A method has been devised whereby the thickness of a duplicate of a biological section can be used as an estimate of the mean thickness of that section, thus enabling the magnitude of the intersectional thickness variation in a series to be assessed and corrected for. In this method the microtome knife cuts simultaneously with the section of biological material a plastic duplicate thereof. Such plastic duplicates are optically homogeneous and their thickness can readily be determined in an interference microscope. The method has been adapted for embedded as well as for frozen tissue. Its value has been checked by making a total of 714 thickness determinations on 51 tissue sections and their duplicates. Analysis of regression showed that when the nominal section thickness indicated by the microtome feed setting was used as an estimate of mean section thickness the coefficient of variation was 13% for paraffin sections and 45% for frozen sections with a nominal thickness of 5 µ. The corresponding figures when thickness duplicates were used as estimates of mean section thickness amounted to merely 7% and 14% respectively.