Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation

Four fuchsin analogues (Pararosaniline, Rosaniline. Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches. Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 microns. Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.     

Standardization and quality controls for the methylated DNA immunoprecipitation technique

MeDIP (Methylated DNA Immunoprecipitation) is a relatively recent technique aimed to enrich the methylated fraction of DNA with an antibody directed against 5-methyl-cytosine. MeDIP processed samples are suitable for investigation of the methylation status of specific genomic loci and for performing genome-wide screening when hybridized to DNA methylation microarrays or analyzed by deep sequencing. Here, we describe a standardization protocol and quality controls to assess the specificity, reproducibility and efficiency of the MeDIP procedure. These may have utility when comparing results between samples and experiments within laboratories and between laboratories.     

The dependance of the absorbance of the final chromophore formed in the Feulgen-Schiff reaction on the pH of the medium

Summary Polyacrylamide model films containing DNP were investigated for the influence of buffer rinses of various pH values and for various periods of treatment after Feulgen-Schiff staining.A citric acid/phosphate buffer of pH 5.6 as the final medium before the dehydration and embedding procedures is recommended to obtain a defined end product with a high molecular absorbance and good stability with time.     

The interaction of apurinic acid aldehyde groups with pararosaniline in the Feulgen-Schiff and related staining procedures.

The equilibrium reactions involved in the formation of the apurinic acid (APA)-Schiff chromophores in the staining phase of the Feulgen-Schiff reaction do not allow a quantitative conversion of APA to these chromophores. By modification of the sulfite and dye concentrations and the pH of the staining reagents, or by using better solvents for pararosaniline like acetic acid or dimethylsulfoxide (DMSO) a shift of these equilibria was attempted in order to obtain a higher amount of APA-bound dye. A 40% higher absorbance, when compared with the normal Schiff-staining, was obtained in model films by staining with a saturated solution of pararosaniline in a 1:1 v/v mixture of DMSO and SO2-water, followed by rinsing in SO2-water. A doubling of the absorbance resulted in the same objects when a saturated solution of pararosaniline in a 2 M acetic acid/acetate buffer of pH 4.45 was used for staining, followed by a short rinse in SO2-water. Amino groups (as found in histones) are shown to compete with the amino groups of pararosaniline for the APA aldehydes. This effect, although causing lower staining intensities, is shown not to be the explanation for the differences in stain content found between more and less compact forms of chromatin. Depending on the pH, and dye and sulfite concentrations of the staining reagents, the following components are considered as possible contributors to the mixture of chromophores (Duijndam et al., 1973 b) formed between APA and Schiff's reagent or its modifications: 1. An acid labile component with a wavelength of maximal absorbance (lambda max) near 510 nm; its structure is probably the azomethine--CH=N--; 2. A relatively acid stable component with a high value of molecular absorbance (epsilon), an lambda max near 570 nm and possibly having an enamine structure--CH=CH--NH--; 3. A component with intermediate acid stability, low epsilon, and lambda max near 540 nm, and which is probably an alkylsulfonic acid --CH(SO3H)--NH--compound. Small differences in the staining conditions in the histochemical application of the Feulgen-Schiff reaction may cause a shift in the ratio between especially components 2 and 3, resulting in variations in stain content and in lambda max.     

Progress in the Standardization of Stains the Standardization of Eosin and Related Dyes

Decolorizers which are distinctly acidic or basic in their chemical nature give abnormally high decolorization in the Gram stain for bacteria. Acidic substances yield more regular results. Ideally an “inert” decolorizer should be used, but ordinarily such substances will not dissolve the dye or dye-mordant precipitate from the smear. The most practical substances seem to be those so very slightly acidic in character as to be practically inert, such as acetone or alcohol, or a mixture of such substances.     

Standardization of the twinning rate

There is a great interest in comparing twinning rates. These comparisons can be performed between different time periods for a specific population, between different regions within the same country, and between different populations. However, there are several factors (maternal age, parity, urbanization, etc.) that influence the twinning rate. The most dominant one is maternal age, and because the age distribution of the mother varies, it is necessary to standardize the data to make these comparisons. If we want to compare the twinning rates in different countries, we have to face the problem that the composition of the data from different countries may differ to a great extent. The applicable method is determined by the data of the lowest quality. Often the available data do not allow the traditional (direct and/or indirect) methods of standardization. Under such circumstances other methods have to be used. Earlier, Fellman and Eriksson (1987) proposed and successfully applied a new method. In this article we discuss the standardization problem in more detail. We suggest different methods and apply them to different data on twinning from Australia, Finland, and Baden-Württemberg (West Germany). The new standardization methods give standardized twinning rates similar to the rates obtained by traditional methods. It is noted that, irrespective of standardization method, changes in maternal age alone cannot explain temporal or regional variations in the twinning rate. Other factors that may raise or lower the twinning rate are decreasing parity, sociodemographic changes with increased communication, which causes the breakup of isolates, and deteriorating physical condition of mothers as a result of increased industrialization and urbanization.     

Standardization of the study of maxillofacial roentgenology. II. Standardization of the methodology of orthopantomography

The main factor of a good quality of orthopantomograms is a correct position of the occlusion plane and chin support, the straightening of the cervical vertebrae, head fixation and a choice of physicochemical conditions for exposure. All these conditions ensure the standardization of orthopantomography, reducing to the minimum errors in investigations.     

Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis.

From a practical point of view, one of the most difficult issues in the standardization of IHC for FFPE tissue is the adverse influence of formalin upon antigenicity, as well as the great variation in fixation/processing procedures. Based on previous study, an additional study using four markers demonstrated the potential for obtaining equivalent IHC staining among FFPE tissue sections with periods of formalin fixation ranging from 6 hr to 30 days. On this basis, the following hypothesis is proposed. "The use of optimized AR protocols permits retrieval of specific proteins (antigens) from FFPE tissues to a defined and reproducible degree (expressed as R%), with reference to the amount of protein present in the original fresh/unfixed tissue". This hypothesis may also be presented mathematically: the protein amount in a fresh cell/tissue, expressed as Pf, produces an IHC signal in fresh tissue of integral(Pf). When the identical IHC staining plus AR treatment is applied to a FFPE tissue section, the IHC signal may be represented as integral (Pffpe). The degree of retrieval after AR (R%) is calculated as follows: R% = integral (Pffpe)/ integral (Pf) x 100%. The amount of protein in the FFPE tissue may then be derived as follows: Pffpe = Pf x R%. In a situation where optimized AR is 100% effective, the IHC signal would then be of equal strength in fresh tissue and FFPE tissue, and Pffpe= Pf. Further studies are designed to test the limitations of the proposed hypothesis.     

Progress in the Standardization of Stains

The Commission must acknowledge the assistance recently given by two commercial concerns in developing two new products, namely, a pure type of hematein and a special grade of acid fuchsin.     

Progress in the Standardization of Stains

Since the original publication of this staining method (Stain Techn., 18, 95), various improvements have been made. These are chiefly in the formula for acid alizarine blue, Solution 2, given below and the composition of Solution 4. The author substituted ammonia alum for aluminum sulphate, and Richard C. Webster of our department did the work with the acetic-acid-sodium-acetate buffer. He found that the final staining solution of pH 2.9 gave the sharpest and bluest nuclei and clearest transparent and contrasting cytoplasm of a pinkish hue. With our previous formula, muscle fibrils often stained so deeply that they detracted from the value of the slides, made them too opaque and obscured the nuclei. This has now been overcome. This also allows the elastic tissue to show better and to contrast with muscle fibers. This is especially noted in studies of the heart and blood vessels.     

Progress in the Standardization of Stains

As a preliminary step in an investigation designed to reveal the coverage of a spray film on conifer needles after various periods of weathering, it was necessary to find a microtechnic which would leave the spray coat unaltered, and yet differentiate it from the underlying tissues. The usual operations of killing and embedding were, of course, not applicable.