Intensity of Feulgen staining of rat liver nuclei fixed with two different concentrations of formalin

Summary The results presented in this paper indicate that following fixation of rat liver in either 40% (w/v) or 10% formalin solution, Feulgen staining is far greater in the tissue fixed in the former fixative as compared with the same fixed in the latter. A possible mode of action of formalin towards fixation and subsequent Feulgen staining is suggested.     

Hot hydrochloric acid hydrolysis and UV Feulgen staining of rat liver sections fixed in CRAF and CRAF-like fixative.

Sections of rat liver fixed in CRAF III and Nawaschin's fixative in Dutt's modification were subjected to hydrolysis in 1N HCl at 60 degrees C for different periods of time and to Schiff's staining according to the UV Feulgen technique. The study showed that Feulgen reaction intensity depends upon time of hydrolysis, optimum coloration being possible only after 10-15 min of hydrolysis. Prolongation of hydrolysis beyond this time produced decreased staining intensity which is retained for further 35 min of hydrolysis thus forming a plateau. Further prolongation of hydrolysis results in gradual deterioration of the staining intensity which culminates in utterly pale coloration of the nuclei after one hour's hydrolysis. A possible explanation for this phenomena is suggested.     

Increase in Feulgen staining intensity by pre-treatment with different reagents.

In the experiments sections of rat tissues fixed in 10 per cent buffered neutral formalin for 3 hour and treated for 15 minutes with different chemical reagents such as pyridine, tributylamine, urea, tris sodium nitrite and sodium hydroxide were subjected to hydrolysis in 6 N HCl at 25 degrees C for 20 minutes and stained by the UV Feulgen technique. The results reveal a far more intense staining of the nuclei in tissues treated with any of the chemicals mentioned than in untreated controls. The possible role of these chemicals in enhancing the staining intensity is discussed.     

Comparative studies of Feulgen hydrolysis for DNA. I. Influence of different fixatives and polyethylene glycols

We compared the Feulgen hydrolysis curves (37 degrees C, 5 M HCl) of human blood lymphocytes fixed by the following four methods: 96% ethanol, 60 min at 20 degrees C; ethanol-acetone, 1:1, 120 min at 4 degrees C; ethanol-glacial acetic acid mixture (3:1), containing 2% of formaldehyde (EAF), 90 min at 20 degrees C; and ethanol-glacial acetic acid (3:1), 60 min followed by 5% chrome alum solution for 360 min at 20 degrees C. The best results were obtained with EAF-fixation, with respect to the highest amount of DNA-Schiff complex at the peak point of the curve, the longest "plateau" region and the smallest scattering of DNA-Schiff amount values along the "plateau". With other types of fixation the addition of polyethylene glycol (PEG) 6,000 to the hydrolysis solution resulted in modification of the shape of the hydrolysis curve so that it became nearly similar to the EAF-curve. The effect of PEG6000 on the EAF-curve was minimal. Slides fixed by ethanol were used for a comparison of polyethylene glycols with m.w. 1,500, 6,000 and 20,000. The longest "plateau" was obtained with PEG6000. The only effect of PEG1500 was a dramatic increase of DNA-Schiff amount at the peak point. PEG20000 had no significant effect on the shape of the hydrolysis curve. The results are discussed in terms of Kjellstrand's "chain with a stable structure" model of Feulgen hydrolysis.     

Reevaluation of optimal Feulgen reaction for automated cytology. Influence of fixatives

The Feulgen reaction is used for cytophotometric quantification of nuclear DNA and texture studies of chromatin structure. It appears that fixative agents are responsible for the microscopic appearance of chromatin. In this investigation, different fixative agents mixed in various proportions were tested for their performance in automated quantitative cytology. It was determined that three factors have to be considered in the choice of a fixative: stain intensity, nuclear area and chromatin texture. In this respect, the Regaud fixative appears to be the best for automatic analysis of Feulgen-stained nuclei.     

Alcian Blue staining of glycosaminoglycans in embryonic material: Effect of different fixatives

Summary Glycosaminoglycans are important components of the extracellular matrix of developing embryos where they are found in the form of proteoglycans. Alcian Blue staining of tissue sections is the technique most commonly used for demonstrating their distribution. Glycosaminoglycans have a high solubility in water, and are easily lost from the tissue during processing, even if non-aqueous fixatives have been used. Formalin and Carnoy's fluid are the most frequently used fixatives, and the addition of cetyl pyridinium chloride has been recommended to reduce glycan solubility.Using sections of day-10 rat embryos containing developing head and heart (both known to be rich in glycosaminoglycans) the effects of ten fixatives have been investigated with and without cetyl pyridinium chloride on the preservation of Alcian Blue-stainable material (at pH 2.5) and tissue structure. The most useful fixatives were Karnovsky's and Sainte-Marie's. Both gave a strong and reproducible staining pattern of the extracellular polyanionic material. Sainte-Marie's gave better preservation of tissue structure, allowing the demonstration of cell-matrix inter-relationships; Karnovsky's gave a better contrast between extracellular and intracellular staining, which is particularly useful at lower magnifications.Cetyl pyridinium chloride is a detergent. Transmission electron microscope observations showed that it causes cell membrane disruption and vesicle formation, which at the light microscopic level, would cause cell membrane-associated glycosaminoglycans to appear as stained strands wholly within the extracellular domain. Therefore the use of cetyl pyridinium chloride is inadvisable where a distinction between surface-related and extracellular glycosaminoglycans is desirable. It has the further disadvantage of enhancing cytoplasmic and nuclear polyanionic material, thus decreasing the differential staining intensity of intracellular and extracellular domains.     

Comparison of Papanicolaou staining and Feulgen staining for automated prescreening

Feulgen staining is considered to be a quantitative DNA-specific cytochemical procedure. The applicability of this staining in high-resolution cytometry was tested in comparison with a regressive Papanicolaou staining. Papanicolaou-stained or Feulgen-stained intermediate and carcinoma cells selected by a cytologist were examined with a Zeiss scanning microscope photometer at 546 and 560 nm, respectively. After cell image segmentation and feature extraction, a statistical data evaluation was carried out by computer. Cell distributions with respect to four selected nuclear features demonstrated the influence of the staining procedure on cell feature measurements. The discriminatory power of the classification system as related to both staining procedures was studied using discriminant analysis. Using only nuclear features, a 7.3% improvement of the overall correct classification rate (from 85.0% to 92.3%) was achieved using Feulgen staining. The misclassification rate was simultaneously reduced by 50%. Using cytoplasmic as well as nuclear features, a 98% rate of correct classification was achieved.     

Feulgen type staining of mammalian tissues with Dahlia-SO2.

The use of the basic dye, Dahlia, which belongs to triphenylmethane group but without a primary amino group in its molecule has been described as useful in the staining of aldehyde groups of acid hydrolysed DNA in tissue sections following the conventional Feulgen procedure. Dahlia-SO2 prepared with sodium hydrosulphite is highly suitable when used at pH 4-0 to 5-0. The absorption characteristics of the stained nuclei indicate on the peak of maximum absorption at 560 nm, whereas, that of the aqueous dye solution is at 590 nm.     

Effects of different fixatives on beta-galactosidase activity.

beta-Galactosidase (beta-Gal) staining is widely used to demonstrate specific gene expression during evaluation of gene targets in vivo. This technique is extremely sensitive to fixation. Optimal fixation conditions are necessary to obtain the maximal beta-Gal activity. In this experiment, Carnoy's and three different aldehyde fixatives were used at different temperatures and over different time points. Kidneys from LacZ-stop-human alkaline phosphatase (ZA/P) double reporter mice were used to generate positive material for the experiment. The results show that glutaraldehyde combinative solution (LacZ) produced the most consistent and reliable results. Paraformaldehyde and formaldehyde were effective as fixatives only at 4C for a period of less than 4 hr, and Carnoy's solution destroyed beta-Gal activity.     

Exposure and removal of stainable groups during Feulgen acid hydrolysis of fixed chromatin at different temperatures

Summary Hyperdiploid Ehrlioh's ascites tumour cells grown in male mice (strain NMRI) were labeled with radioactive nucleotides. The nucleic acids were extracted from fixed, air-dried smears by fractionated hydrolysis and their radioactivity measured by liquid scintillation. The experiments showed that the exposure of aldehydes through removal of purine bases and the elimination of these aldehydes through depolymerisation of DNA were the two main processes responsible for the Feulgen hydrolysis curve. They were shown to be independent and overlapping. The depurination can be described as a simple hydrolytic reaction, while the extraction of DNA depends on a number of different factors. This entails that, in the Feulgen acid hydrolysis procedure, the part of DNA measured is dependent upon the stability of the chromatin. It was found that it is possible accurately to determine the depolymerisation process and thereby roughly correct the measured amount of Feulgen DNA.     

Evaluation of nine different fixatives

Summary An artificial substrate was developed for quantitative testing of the ability of various fixatives to preserve the reactivity of IgG and IgA isotypes ( and chains) and the secretory component (SC) of secretory IgA as model antigens. Polymerized normal rabbit serum was used as matrix and defined amounts (10–0.1 g/l) of antigen were incorporated into it by diffusion before fixation and paraffin embedding. The various fixatives comprised alcohol, routine formalin, glutaraldehyde(1%)-formalin, Baker's formol calcium, formol sublimate, acetic acid(2%)-formol saline, Bouin's fluid, Susa fixative, and carbodiimide. The detection sensitivity afforded by these fixatives was defined as the immunofluorescence staining end point. Compared to the reference value obtained with alcohol ( and chains, 0.06 g/l of IgG and IgA; SC, 0.12 g/l of colostral IgA), an antigen concentration at least 8 times higher was necessary for detection with most of the cross-linking fixatives. Bouin's and Susa fixatives were peculiar in that they required more than 150 times higher antigen concentration for detection of IgG but only 3–8 times higher for IgA. The determined sensitivities were compared with the immunofluorescence performance results obtained on human tissues prepared with the same fixatives; excepting carbodiimide (which produced unacceptable autofluorescence of the substrate matrix) a remarkably good correlation was found with regard to IgG- and IgA-producing cells (especially of the former isotype) and secretory epithelium (IgA and SC). However, the latter result depended on pronase treatment of the tissue sections to unmask epithelial antigens. It was concluded that detection sensitivity as determined with this artifical substrate may give a good idea about the immuno-histochemical performance obtained on tissue prepared with the actual fixative; but the degree of antigenic masking in the various tissue compartments has to be taken into account if the comparisons are to be meaningful.This study was supported by the Norwegian Cancer Society, The Norwegian Research Council for Science and the Humanities, and Anders Jahre's Fund. The results were in part presented at the Symposium on Immunocytochemistry of Lymphomas, Micro 82 Conference, The Royal Microscopical Society, London, July 1982 (Histochem. J. 15:655–689, 1983)     

A method for combined gross skeletal staining and Feulgen staining of embryonic chick tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

Acid hydrolysis characteristics of spleen colony cells under Feulgen staining

A cytophotometrical determination of DNA content was performed in cells of murine spleen colonies originating from bone marrow and in lymphocytes of axillary lymph nodes under various temperatures (22, 25 and 37 degrees C) of hydrolysis (5N HCl). It is shown that acid hydrolysis at 20 and 25 degrees C is most-preferable for proliferated cells of spleen clones and for non-proliferated lymphocytes. It is concluded that hydrolysis curves for clonal cell nuclei in different phases of mitotic cycle practically coincided.