On quantitative histo- and cytochemistry and general reflections

Reproduced, with modification, from Carlsberg Res. Commun. 49, 255–258 by kind permission of the Editor at Springer-Verlag.     

A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.     

Trends in quantification in histochemistry and cytochemistry

Conclusion In this limited review, every relevant technique and many personal credits could not be included, but it is apparent that there has been a steady increase in the armamentarium of applicable techniques for quantification, each with its special uses and limitations. As the trend continues, and beyond the more obvious future which will see consolidation and extension of existing experimental approaches with an increasing use of automation and computerization, we can only guess at what new departures and remarkable innovations will emerge to provide leaps forward toward the goal—the goal of combining localization with quantitation of biological substances and activitiesin situ to reveal the chemistry, and from it the function, of the cell, its substructures and products, in the normal and altered living state.No one would disagree with Cournand who said in the concluding remarks of his 1956 Nobel Prize lecture on the pulmonary circulation, Now what of the future? Perhaps the only incontestable prophecy that can be made is that advances in methodology and advances in understanding go hand in hand. What we can also predict is that the quest of our goal will continue to hold fascination and excitement for those lucky enough to be involved in it.     

A quantitative evaluation of peroxidase inhibitors for tyramide signal amplification mediated cytochemistry and histochemistry

Many peroxidase inhibitors have been used in horseradish peroxidase (HRP) mediated immunostaining and in situ hybridization to quench background peroxidase activity. However, the efficacy of these inhibitors has been controversial, partially due to the lack of a quantitative study. Tyramide signal amplification (TSA) is much more sensitive than other HRP-mediated methods but its super-sensitivity also demands effective inhibition of background peroxidase activity. In searching for an effective peroxidase inhibitor, we have systematically evaluated the efficacy of several peroxidase inhibitors by quantifying the fluorescence intensity in cultured fibroblasts and tissue sections treated with the inhibitors. For cultured cells, 0.05 mM of phenylhydrazine and 1 unit/ml of glucose oxidase gave only moderate inhibition of HRP activity while 1 mM of sodium azide (NaN₃), 3% of hydrogen peroxide (H₂O₂), NaN₃/H₂O₂ combined and 0.02 N hydrochloric acid (HCl) provided more complete inhibition. However, the inhibitory effect of NaN₃/H₂O₂ is reversible upon removal of the inhibitors and followed by incubation and wash to mimic antibody interactions. Similar results were obtained from rat skin wound tissues that have strong endogenous peroxidase activity. Our results recommend the use of HCl and caution the use of phenylhydrazine, glucose oxidase, NaN₃ and H₂O₂ as potent peroxidase inhibitors.     

Microbiological assay for quantitative determination of polyoxin B

Polyoxin B, an antifungal agro-antibiotic, is used for treating serious plant diseases. Until now there have been no reports on the antibiotic quantification in liquid in spite of its need for fermentation studies. This work reported a microbiological assay, the cylinder-plate method, for the determination of polyoxin B. The results of assay were treated statistically by analysis of variance (ANOVA) and were found linear in the range of 10–500 μg/ml (r² = 0.998), precise (intra-assay: relative standard deviation (R.S.D.) = 0.64; inter-assay: R.S.D. = 1.70) and accurate. This newly established method was applied to determine the antibiotic titer in Streptomyces cacaoi fermentation with comparison to HPLC analysis. The microbiological assay was satisfactory for polyoxin B quantification, and a simple, sensitive, cost-effective and specific agar diffusion bioassay for polyoxin B was thus developed. This work also demonstrated the usefulness of the microbiological assay for quantitative analysis of antibiotics in fermentation.     

Intermediate electron-acceptors in quantitative cytochemistry

Summary The efficacy of Meldola Blue (MB), a new intermediate electronacceptor, has been compared with that of phenazine methosulphate (PMS) in the assay of oxidoreductase activity in cryostat sections; various tetrazolium salts have been used as the final electron-acceptors. Three enzymes: succinate dehydrogenase, glucose 6-phosphate dehydrogenase and lactate dehydrogenase were investigated, the activity in sections being quantitated by scanning and integrating microdensitometry. Phenazine methosulphate was superior to Meldola Blue in transferring reducing equivalents from reduced coenzyme to all the tetrazolium salts examined.     

Studies in lipid histochemistry

Summary The control reaction with Schiff's reagent demonstrates besides free aldehydes also plasmalogens and is not a proper control test in lipid histochemistry in cases when reactions with Schiff's reagent are used in tissues containing plasmalogens. For the elimination of genuine aldehydes neutral blocking tests should be applied whenever plasmalogens are present.     

Studies in lipid histochemistry

Summary The histochemical value of fractionated histochromatographic examination of lipids according to Holczabek's (1969) method which gives very good results as regards chromatography, is limited by that phospholipids are not retained quantitatively in sites of their original localization in sections during the first developing in a mixture of petrolether:ether:acetic acid. The staining of phospholipids performed after the first developing therefore does not reveal the localization pattern of all phospholipids.     

Studies in lipid histochemistry

Summary The effect of bromination (carried out especially with potassium tribromide and with bromine water) on various chemical groups in lipids and nonlipidic substances in tissue sections and in spot tests was investigated. Bromination blocks rapidly and effectively the reaction of double bonds with osmium tetroxide in unsaturated fatty acids, in phosphoglycerides, and nonpolar lipids, whereas in sphingolipids some residual staining persists even when the blocking period is prolonged. Similar results were obtained in the case of the UV-Schiff reaction. The vinylether bond in plasmalogens is blocked completely but not so rapidly.Besides double bonds bromination blocks the pseudoplasmal reaction in unsaturated lipids and the reaction of higher fatty aldehydes with Schiffs reagent and with dinitrophenyl hydrazine. Moreover it oxidizes the hydroxy group of cholesterol to a ketoderivative and in the case of ceramide mono- and dihexosides increases the intensity of the PAS reaction.The influence of bromination on various protein groups and the extraction of some loosely bound mucosubstances were demonstrated. The practical implication of these observations in applied lipid histochemistry is discussed.     

Studies in lipid histochemistry

Summary It was shown by experiments with staining reactions performed in situ in blood smears of adults and children, in sections prepared by various procedures, in spot tests, and by chromatographic examination of extracted lipids that haemoglobin is responsible entirely for the staining of red blood cells in the acid haematein test and for the greater part for the staining in the OTAN reaction. The acid haematein test is from the point of view of lipid histochemistry neither specific nor sensitive. On the other hand the OTAN reaction is sensitive although it is not specific. It is concluded that the extraction test with chloroform-methanol must always be performed when the lipidic nature of the demonstrated substance(s) is to be proved unequivocally and that the non-lipidic nature of the residual staining must be always considered.     

Studies in lipid histochemistry

Summary The influence of different terminal rinsing bathes on the results of methods using Schiff's reagent (S.r.) was studied in tissue sections and in chromatograms. The compound resulting in the reaction of aldehydic groups with S.r. is not stable in the acidic pH range. The amount of aldehydic groups which are set free increases with the acidity of the rinsing bath. This results in a substantial weakening of the original coloration. The use of a slightly acid bisulphite-HCl bath followed by thorough washing in tap water proved to be very good in practice. Strongly acidic bathes (2 N-3 N HCl) must be avoided.