Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Staining of interphase nuclei and mitotic figures in cultured cells with Alcian blue 8GX

Summary Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction or Heidenhain iron hematoxylin but without the latters’ length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Metallic Lakes of the oxazines (Gallamin Blue, Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2-3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3-5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

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.     

Histochemical evaluation of glycosaminoglycan deposition in the skin

Histologic demonstration of glycosaminoglycan (GAG) deposition in the skin has been based on the use of either colloidal iron or alcian blue. To define the best technique for the determination of skin GAG content we undertook a prospective study comparing the two stains and evaluating the use of cetylpyridinium chloride (CPC) to enhance fixation. Slides were prepared from skin biopsies obtained from five patients with cutaneous mucinoses. The preparations were coded and examined by three observers. Colloidal iron staining gave a higher intensity for GAG deposits in papillary and reticular dermis. Digestion by specific enzymes identified similar GAGs with either colloidal iron, or alcian blue; however, colloidal iron made GAGs more obvious, partly due to the contrast afforded by the yellow background stain. The addition of CPC to the fixative appreciably enhanced GAG fixation without interfering with the action of enzymes. Experimentally, we confirmed this effect of CPC by determining a pronounced decrease in GAG leakage into the fixative from CPC treated human umbilical cord. We conclude that the combination of CPC fixation and colloidal iron staining gives the best definition of skin GAGs in clinical specimens.     

Dichromate-Chlorate Perfusion Prior to Staining Degeneration in Brain and Spinal Cord

A method of fixation compatible with both the Nauta-Gygax and Swank-Davenport procedures for degenerating nerve fibers, which shortens the time required by the former procedure, is as follows: The central nervous system is perfused with a 0.9% aqueous solution of NaCl followed by an aqueous solution containing 5% K₂Cr₂O₇ and 2.5% KClO₃. The central nervous system is then hardened in 10% formalin for 1-3 days. Tissue for Marchi-type staining can be taken at this stage. For silver staining, the processing is continued by immersion overnight in 10% formalin in 20% alcohol, and frozen sections cut the next day. Sections, up to 50μ in thickness, are collected in 10% formalin and impregnated by the Nauta-Gygax technique. Best results are obtained by impregnating within 24-48 hr after sectioning.     

A Combination Verhoeffs Elastic and Masson's Trichrome Stain for Routine Histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeffs technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

A combination Verhoeff's elastic and Masson's trichrome stain for routine histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeff's technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

Immunoperoxidase detection of myeloid antigens in glycolmethacrylate-embedded human bone marrow

Different methods for fixation and exposure of antigenic determinants were tested for detection of a granulocytic differentiation antigen by the monoclonal antibody L12-2, using an indirect immunoperoxidase method on semi-thin sections of undecalcified, glycolmethacrylate-embedded human bone marrow biopsies. Fixation in Bouin's solution for 3 hr gave a more intense and more homogeneous immunological staining than fixation in absolute methanol, 4% formalin, B5, or Michel's medium, and the morphological detail was excellent. Digestion by pronase or trypsin was required. Coating the glass slides with Alcian blue prevented loss of sections from the slides during the staining procedure. Bouin fixation also made possible detection of two other differentiation antigens expressed in the granulocytic series, using the monoclonal antibodies 1G10 and R1B19. Furthermore, the same technique also permitted detection of factor VIII-RAg in the megakaryocytes, as well as recognition of cells of the erythroid series by use of polyclonal rabbit antisera.     

An Easily Controlled Regressive Trichromic Staining Method

Three modifications of Mallory's connective tissue stain are described and some features of the action of picric acid are discussed.

In the first and most critical method the nuclei are stained in an iron hematoxylin and then differentiated in a picric acid solution containing orange G. This not only differentiates the nuclei, but stains all other elements yellow. The section is then washed in running water to remove the yellow color from all tissues except those which are to remain yellow in the final preparation (usually the erythrocytes). The section is next stained in an acid fuchsin mixture and then differentiated until the desired depth and contrast is obtained. Staining in anilin blue follows and this in turn is differentiated to suit. The section is then dehydrated and mounted.

In the second method the nuclei are stained in hemalum (e.g. Harris's) for a short time; the section is then rinsed and immersed in a mixture of picric acid and acid fuchsin and thereafter is differentiated; it is next passed into anilin blue w. s. and then differentiated and mounted as before. This is less critical than method I, but can be applied to large batches of slides at a time.

The third method is a one-solution method. After staining the nuclei in hemalum, the section is immersed in the “Picro-Mallory” solution, differentiated briefly, dehydrated and mounted. This modification, while being the least critical, is most suitable for routine use when the tissues have been fixed in a fluid containing chromate; the other commonly used fixatives, while giving useful results, are not so good.     

Metallic Lakes of the oxazines (Gallamin Blue,Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2–3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3–5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

A Cresyl Fast Violet Stain for Bacteria and Fungi in Tissue

The cresyl fast violet staining method was modified to eliminate differentiation. Paraffin sections from tissues fixed in Zenker-formol were stained in a 1% aqueous solution of cresyl fast violet (Chroma), adjusted to pH 3.7 with acetic acid, washed in running tap water, dehydrated and covered. Because basophilia increases with time of fixation or storage in formalin or Kaiserling's fluid, dilution of the dye solution to 0.5-0.1% is recommended for such material. Bacteria, nuclei, Nissl substance, and lipofuscin were colored dark blue; fungi, blue to purple; and cytoplasm and muscle fibers, light blue. Collagen and reticulum fibers were only faintly stained. Thus, microorganisms were easily visible against the lightly colored background. In formalin-fixed material, bile pigment was colored olive green. Because this method does not require differentiation, it gave uniform results even in the hands of different users. Little or no fading was observed in sections stored for more than 2 yr.     

Phase-partition fixation and staining of Drosophila eggs.

Aqueous solutions of alcohol-acetic acid-formalin or glutaraldehyde-acrolein were shaken with heptane and heptane phase used for fixation. Phase-partition fixation is akin to fixation with vapor. The organic solvent, immiscible with water, penetrates hydrophobic membranes and carries the fixative in contact with water phase of the tissue. Only the fixative enters the tissue, without changing the ionic and water-soluble substance concentrations in the tissue. The quality of this fixation for optical or electron microscopy was as good as that of any conventional fixation method. Staining with basic fuchsin after 2 N HCl hydrolysis gave brilliant staining of nuclei, more intense than that with Feulgen reagent, while cytoplasm remained nearly colorless. Fixing and staining procedures for Drosophila eggs are given.     

Applications of fluorochromes to pollen biology. II. The DNA probes ethidium bromide and Hoechst 33258 in conjunction with the callose-specific aniline blue fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

Applications of Fluorochjromes to Pollen Biology. Ii. The Dna Probes Ethidium Bromide and Hoechst 33258 in Conjunction with the Callose-Specific Aniline Blue Fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

Combined in situ zymography, immunofluorescence, and staining of iron oxide particles in paraffin-embedded, zinc-fixed tissue sections

Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

Combined In Situ Zymography, Immunofluorescence, And Staining Of Iron Oxide Particles In Paraffin-embedded, Zinc-fixed Tissue Sections

AbstractSuperparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips

A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1–10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G₁/S interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 M). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of 1×10⁵/root and 1.4×10⁵/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.     

A simple and accurate method for the measurement of the growth of cell monolayers

Summary A procedure has been developed for the accurate, quick, and simple indexing of the growth of insect cell monolayers with a reticule in the eyepiece of an inverted phasecontrast microscope. A magnification of ×500 is desirable for accuracy and ease of counting. Cell nuclei whose circumferences include any of the 25 points in the reticule are enumerated in each microscopic field. Counts of as few as five fields gave an error of 18.8% per flask. Ten fields gave an error of 11.5% and provided a sufficiently accurate comparison for many purposes. Forty counts allowed the measurement of differences between treatments with an error of 5.85%. A large number of treatments can be handled simultaneously, due to the small number of replicates necessary and the small amount of time required per treatment. When the capacity of different batches of lactalbumin hydrolysate to support growth was tested in our insect tissue culture medium, some of them were found to be suitable and others unsuitable. Doubling time of cell populations was measured, after the ratio of the average area of the nuclei at time 0 to that at timeX was used to multiply the number of nuclei counted at timeX. Average nuclear areas were satisfactorily measured by simple measurements of nuclear diameter on an ocular micrometer. The cell nuclei tended to decrease in area when cell monolayers reached confluence or became crowded. The number of replicates required was reduced, because the same flask could be used several times without disturbing the cell monolayer. The method of counting nuclei in a monolayer by means of reticule points and a phase-contrast microscope can also be adapted to the estimation of the absolute number of cells growing on the bottom of a flask. Portion of a thesis by the senior author submitted for the Ph.D. degree to the Graduate College of the University of Illinois. This research was supported in part by grant GB 20915 from the National Science Foundation and United States Public Health Service grant AI 6392 from the National Institutes of Health.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.