STUDIES ON NUCLEIC ACID METACHROMASY : I. The Effect of Certain Fixatives on the Dye Stacking Properties of Nucleic Acids in Solution

The stacking coefficients (K's) of nucleic acids have been thought to influence the color contrast between DNA and RNA in tissue sections stained with metachromatic dyes. This idea was tested by titrating toluidine blue (TB) and acridine orange (AO) in solution against DNA and RNA, native or treated with formaldehyde, acrolein, or Carnoy's fluid. Absorption spectra at varying polymer-dye ratios were used to compute K values by the methods of Bradley and colleagues. Results with both dyes fit Bradley's stacking equations. Fixatives did not block dye-binding sites but markedly altered K values. K of DNA was low, unaffected by aldehyde fixative, increased by Carnoy's fluid or heat denaturation. K of RNA was higher than that of DNA and was increased greatly by formaldehyde, almost as much by acrolein, considerably less by Carnoy's fluid. Aldehyde effects were partially reversed upon removal of aldehyde by dialysis. These observations accord with known effects of aldehydes and denaturation upon nucleic acid conformation. Differences between K's of DNA and RNA were greater after aldehyde treatment than after Carnoy's, and were greater with AO than with TB. This is generally consistent with the magnitude of the color contrasts observed in tissues. Additional factors must contribute to the intense color contrast observed in acrolein-fixed tissues stained with TB.     

Romanowsky-type staining: Enzymatic analysis of nuclear metachromasia in formalin-fixed paraffin sections

The red color of nuclei produced in formol-fixed paraffin sections stained with toluidine blue has been investigated by using deoxyribonuclease (DNase), ribonuclease (RNase) and 0.1 M Tris buffer. The action of DNase on formol-fixed material is not fully reliable, but clear-cut when positive. Nuclear basophilia and metachromasia is removed, nucleolar and cytoplasmic RNA is preserved. The picture produced by RNase depends to some extent on the concentration and acidity of the toluidine blue used for subsequent staining. Cytoplasmic RNA is always removed, while the red stain in nuclei usually remains intact. With 0.1% toluidine blue in 1% acetic acid, a nuclear color change from red to pale green is observed. Using this same staining solution, it can be shown that 0.1 M Tris buffer (overnight extraction at 37° C) will remove cytoplasmic RNA but leave intact the nuclear material that stains red. A red to green shift can subsequently be produced by RNase. From this it is deduced that there is a chromatin-associated nuclear RNA fraction which can be removed by the enzyme, but is stable to the buffer solution.     

Staining Paraffin Embedded Sections of Scald of Barley before Paraffin Removal

Staining of paraffin embedded sections with periodic acid-Schiff reagent and fast green before paraffin removal resulted in differentiation of barley seed and leaf tissue from fungal structures of Rhynchosporium secalis. Crystal violet, toluidine blue O and aniline blue also successfully stained fungal structures of R. secalis in barley leaf tissues. Staining of embedded sections before paraffin removal allows simple processing of a series of sections, saves time and reduces solvent consumption.     

Combined Bright Field and Fluorescence Staining of Paraffin Sections for Studying the Fertilization Process in Plants

PAS-toluidine blue O—aniline blue staining of paraffin sections allows study of histological and cytological detail while retaining aniline blue induced fluorescence in all “callose sites”. Because most autofluorescence is eliminated by the PAS-toluidine blue prestaining, the detail and contrast of the fluorescence image is superior to slides stained in aniline blue alone. Slides are stained by the PAS reaction, 0.03% toluidine blue O, alkaline 0.005% aniline blue, and mounted directly in aqueous mounting medium.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

Optimization of a histopathological biomarker for sphingomyelin accumulation in acid sphingomyelinase deficiency

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy.     

Direct Staining of Reticular Fibers with Gold Chloride

Reticular fibers are selectively stained in paraffin sections of formalin-fixed or Bouin's-fixed tissue as follows: 1% aqueous solution of gold chloride for 20 min, followed by a 10 min immersion in an aqueous solution containing 5% Na₂CO₃ and 0.5% KOH. The sections then are placed in a 5% aqueous solution of KI for 2 min. Counterstaining with a 0.25% aqueous solution of methylene blue chloride is optional. The reticular fibers stain dark pink; the collagen bundles are a light pink to straw color without the counterstain, or a light blue color when the methylene blue is used.     

Indole reactions of mast cells and enterochromaffin cells related to fixations with and without aldehydes

Summary Survey of a considerable number of rat, mouse and hog tissues which presented large numbers of mast cells in preparations stained with toluidine blue and other metachromatic or basic dyes at low pH levels, revealed numbers of oval bodies of about the same size as mast cells which reacted weakly or even moderately to the postcoupled benzylidene indole reaction. The numbers of these were always less than that of mast cells in toluidine blue sections of the same blocks. They never occurred in clusters of perhaps 15–20 in a single high power field, as mast cells often do. Smooth and especially striated muscle which often formed the background tissue where most mast cells are found with metachromatic stains, regularly present indole reactions due to protein tryptophan. This is usually equal to or stronger than that in the supposed mast cells.Indole reactive bodies whose morphology suggests mast cells are also present in similar numbers in formaldehyde and glutaraldehyde fixed tissue as well as with aldehyde free fixations. Glutaraldehyde and formaldehyde are known to inhibit the benzylidene reaction of 5-HT in vitro (30 min for glutaraldehyde, 3 h for formaldehyde) (Lillie, 1977). This action was avoided in mercury and lead heavy metal fixations and in acetone, Carnoy, chloroform methanol and similar fixations.The mast cell-like bodies are best explained as tangential or oblique sections of individual muscle fibers. We have described the same phenomenon with the ferric ferricyanide (Golodetz-Unna, 1909) reaction (Lillie et al., 1978a), and the PCB reaction is that of tryptophan in these muscle cell sections.In contrast to the DMAB type reaction failure acid diazosafranin successfully demonstrated mast cells with both aldehyde and aldehyde free fixations. This reaction has been shown to occur with 5-HT and 5-HTP (Lillie et al., 1973).     

Staining of Different Tissues in Thick Epoxy Resin-Impregnated Sections of Human Fetuses

Sections of undecalcified human fetuses, fixed in formaldehyde, embedded in the epoxy resin Biodur E 12 and cut on a diamond-wire saw were stained according to a slight modification of the method described by Laczko and Levai. The sections were immersed in a methylene blue/azure II solution at 90 C for at least 3 min and counterstained with a basic fuchsin solution at the same temperature. Differential staining was as follows: bone stained pinkish; cartilage, violet; collagen fibers, blue-violet; elastic fibers, red and muscle fibers, green-blue. Most other tissues were stained blue-violet against the transparent background of the embedding epoxy resin. Thanks to the distinct and differential staining of each tissue, contrast is sufficient for black and white as well as for color photography.     

Staining of different tissues in thick epoxy resin-impregnated sections of human fetuses

Sections of undecalcified human fetuses, fixed in formaldehyde, embedded in the epoxy resin Biodur E 12 and cut on a diamond-wire saw were stained according to a slight modification of the method described by Laczkó and Lévai. The sections were immersed in a methylene blue/azure II solution at 90 C for at least 3 min and counterstained with a basic fuchsin solution at the same temperature. Differential staining was as follows: bone stained pinkish; cartilage, violet; collagen fibers, blue-violet; elastic fibers, red and muscle fibers, green-blue. Most other tissues were stained blue-violet against the transparent background of the embedding epoxy resin. Thanks to the distinct and differential staining of each tissue, contrast is sufficient for black and white as well as for color photography.     

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.     

Histochemical Applications of Two Phenanthridinium Compounds

The fluorescent compounds ethidium monoazide and ethidium bromide were found to react intensely with nucleic acids of fixed, paraffin embedded tissues of rat and mouse. For routine staining, 10^-5 M solutions of ethidium bromide and its monoazide analogue were virtually identical in their reactions. Fresh frozen sections of the tissues reacted in the same manner as fixed, paraffin embedded samples. Fluorescence of DNA and RNA in rat pancreas could be selectively abolished by taking advantage of the greater sensitivity of RNA to acid hydrolysis. Hydrolysis in aqueous solutions (1 N HCl at 55-60 C) abolished RNA fluorescence in 5 min, whereas 20 min or longer were required to destroy DNA fluorescence. DNA fluorescence was selectively abolished by 3 hr in 0.1 N HCl in anhydrous methanol while the RNA remained unaffected. Rat pancreas stained with the 10^-5 M ethidium compounds below pH 5.0 showed reduced RNA fluorescence, but the DNA continued to fluoresce brightly at pH 0.6. Reducing the pH of the staining solution to pH 1.0, therefore, was an additional method of selectively abolishing RNA fluorescence. Ethidium solutions in 5.0 M NaCl at pH 5.0 had little effect on DNA or RNA fluorescence. This new method of examining nucleic acids in fixed tissue samples opens new approaches to the histochemistry of these substances. The method also offers new possibilities for the study of mutagenic drug-DNA interactions.     

Quantitative microspectrophotometry of RNA in plant tissue

Synopsis Chemical estimation of nucleic acid, essentially RNA, in fixed tissue from Jerusalem artichoke tubers, coupled with an examination of the types of RNA in the fixed tissue by gel electrophoresis, demonstrates that ribosomal and soluble RNA are preserved in this tissue after various fixation procedures including methanol, ethanol-acetic acid and aqueous formaldehyde. Tissue fixed in ethanol-acetic acid or formaldehyde is resistant to loss of nucleic acid by aqueous extraction but tissue fixed in all three standard fixatives loses nucleic acid in citrate buffer under conditions used for Azure B staining. The presence of Azure B in the buffer does not wholly prevent this loss. Tissue fixed in formaldehyde or mixed fixatives containing formaldehyde is resistant to loss of nucleic acid during treatment with EDTA to obtain cell suspensions.Preliminary experiments with Azure B, Gallocyanin chrome-alum and Methylene Blue showed that the Gallocyanin technique is the most practicable for demonstrating RNA cytochemically. Its specificity was confirmed by ribonuclease extraction of the tissue. Optimum staining conditions, requiring treatment of the tissue in Gallocyanin chrome-alum solution overnight at 40°C, are established for the artichoke tissue and for paraffin sections of pea shoot apices.     

Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy.

In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 microns in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylin-eosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

A Simplified Stain for Hemoglobin in Tissues Or Smears Using Patent Blue

The following technic, based on the patent blue V hemoglobin reaction, is useful for identifying hemoglobin in tissue fixed in neutral formaldehyde solution and embedded in paraffin:

Stain the deparaffinized, hydrated sections 3 to 5 minutes in the working reagent, prepared by adding 2 ml. of glacial acetic acid and 1 ml. of 3% hydrogen peroxide to 10 ml. of the filtered stock solution (1 g. patent blue, 10 g. zinc powder, and 2 ml. glacial acetic acid). Counterstain 30 to 60 seconds in 1:1000 safranin solution in 1% acetic acid, rinse, dehydrate with alcohols, clear in xylene and mount in clarite. Total time required, 37 minutes.

Blood and tissue and smears may be stained, following fixation in methyl alcohol, by applying the working reagent as above.     

Staining of different endocrine cells with hydrochloric acid-toluidine blue in Epon embedded rat tissue

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 hr in 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.     

Staining of Different Endocrine Cells with Hydrochloric Acid-Toluidine Blue in Epon Embedded Rat Tissue

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 turn 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.     

A Histochemical Examination of the Staining of Kainate-Induced Neuronal Degeneration by Anionic Dyes

Anionic dyes, notably acid fuchsine, strongly stain the nuclei and cytoplasm of neurons severely damaged by injury or disease. We provide detailed instructions for staining nervous tissue with toluidine blue and acid fuchsine for optimal demonstration of injured neurons. Degeneration was induced in the hippocampus of the mouse by systemic administration of kainic acid, and the resulting acidophilia was investigated using paraffin sections of the Carnoy-or Bouin-fixed brains. The affected cells were bright red with the toluidine blue-acid fuchsine sequence. Their nuclei were stainable also with alkaline Biebrich scarlet and with the 1,2-naphthoquinone-4-sulfonic acid-Ba(OH)₂ method; all staining was blocked by benzil but was relatively refractory to deamination by HNO₂. These properties indicated an arginine-rich protein. The nuclei were strongly acidophilic in the presence of a high concentration of DNA (strong Feulgen reaction), and acidophilia could not be induced in normal neuronal nuclei by chemical extraction of nucleic acids. The cytoplasmic acidophilia of degenerating hippocampal neurons was due to a protein rich in lysine (extinguished by alkalinity, easily prevented by deamination, and unaffected by benzil). Stainable RNA was absent from the perikarya of the affected cells, but normal neuronal cytoplasm did not become acidophilic after extraction of nucleic acids. We suggest that kainate-induced cell death is preceded by increased production of basic proteins, which become concentrated in the nucleus and perikaryon. Groups of small, darkly staining neurons were seen in the cerebral cortex in control and kainite-treated mice. These shrunken cells were purple with the toluidine blue-acid fuchsine stain, and were attributed to local injury incurred during removal of the unfixed brain.     

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. II. Practical applications for light microscopy

The application of the new nucleic acid dye Cuprolinic Blue to cell smears and tissue sections has been described. Without added cations, Cuprolinic Blue stains both DNA and RNA, whereas in the presence of 1 M MgCl2, Cuprolinic Blue specifically stains single-stranded RNA only. The total RNA can be stained after removal of DNA by DNAase digestion. Fixation in a modified Carnoy solution gave optimal staining results in all cases tested. By cytophotometry, a reliable and reproducible relative estimate can be obtained of the total nucleic acid content, the total RNA content and the amount of single-stranded RNA alone per cell.