Hematoxylin substitutes: fluorone black and methyl fluorone black (9-phenyl- and 9-methyl-2,3,7-trihydroxy-6-fluorone) as metachrome iron alum mordant dyes.

The phenyl and methyl trihydroxyfluorones, hitherto used histologically only in the rather difficult and unreliable Turchini technics for discriminating deoxyribonucleic from ribonucleic acid, find a new use as iron mordant metachrome dyes which act as nuclear stains. Nuclear staining is unaffected by acid extraction of nucleic acids, as with hematoxylin lakes. The two dyes, named by Liebermann and Lindenbaum 9-phenyl-2, 3, 7-trihydroxy-6-fluorone, have also acquired (illustrating with the phenyl homolog) longer chemical names of the form 2,6,7-trihydroxy-9-phenylisoxanthene-3-one (Eastman). Aldrich and Pfalz-Bauer adhere to the Liebermann-Lindenbaum nomenclature. The trivial name fluorone black is proposed for the phenyl homolog and methyl fluorone black for the methyl homolog. The iron lake of fluorone black appears to be a useful substitute for iron hematoxylin, methyl flurone black less useful. Neither dye has the diverse capability of hematoxylin.     

Hematoxylin Substitutes: Fluorone Black and Methyl Fluorone Black (9-Phenyl- and 9-Methyl-2, 3, 7-Trihydroxy-6-Fluorone) as Metachrome Iron Alum Mordant Dyes

The phenyl and methyl trihydroxyfluorones, hitherto used histologically only in the rather difficult and unreliable Turchini tecbnics for discriminating deoxyribonucleic from ribonucleic acid, find a new use as iron mordant metachrome dyes which act as nuclear stains. Nuclear staining is unaffected by acid extraction of nudeic acids, as with hematoxylin lakes.

The two dyes, named by Liebermann and Lindenbanm 9-phenyl-2, 3, 7-trihydroxy-6-fluorone and 9-methyl-2, 3, 7-trihydroxy-6-Ruorone, have also acquired (illustrating with the phenyl homolog) longer chemical names of the form 2, 6, 7-trihydroxy-9-phenylisoxanthene-3-one (Eastman). Aldrich and Pfalz-Bauer adhere to the Liebermann-Lindenbaum nomenclature. The trivial name fluorone black is proposed for the phenyl homolog and methyl fluorone black for the methyl homolog.

The iron lake of fluorone black appears to be a useful substitute for iron hematoxylin, methyl fluorone black less useful. Neither dye has the diverse capability of hematoxylin.

Aided by a contract from the National Cancer Institute NO-1-CB-43912     

Differential Staining with a Mixture of Safranin and Fast Green FCF

Mounted deparaffinized sections were stained for 30-60 minutes at room temperature in a mixture of equal volumes of 0.1% aqueous solutions of safranin O and fast green FCF filtered before use. They were then washed in distilled water for 5 minutes, blotted, washed in 2 changes of absolute alcohol (2-3 min) and mounted from xylene. The nucleic acids are stained purplish-red, half esters of sulfuric acid orange, and proteins green. The procedure is applicable to a variety of materials fixed in a number of reagents though best results are obtained after acetic-alcohol fixation. Bouin's fluid and 10% neutral formalin are not suitable fixatives for this procedure. After acetic-alcohol fixation, the staining procedure may be used in conjunction with enzyme or extraction technics in order to characterize certain chemical components of cells or tissues. The safranin-fast-green technic has proved useful in investigations of pathological changes in tissues; in the visualization of secretory granules and in studies of cellular differentiation. The technic also would appear to greatly facilitate mitotic index determinations.     

A modification of the tannic acid-phosphomolybdic acid-dye stain for demonstrating myoepithelial cells in formalin fixed tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin sections are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:1 methanol:acetic acid, rinsed in 9:1 methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

Cytochemical evaluation of the Guard procedure a regressive staining method for demonstrating chromosomal basic proteins. I. Effects of fixation, blocking reactions, selective extractions, and polyacid "differentiation".

Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for "sex chromatin" display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are "differentiated" in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, "condensed" chromatin can be stained preferentially. Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 - probably due to extraction of some histone fractions-and the least amount of dye was bound in Bouin's-fixed chromatin - probably due to blockage of arginine residues by picric acid. The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl. The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored.     

A Modification of the Tannic Acid Phosphomolybdic Acid-Dye Stain for Demonstrating Myoepithelial Cells in Formalin Fixed Tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin section are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:l methanol:acetic acid, rinsed in 9:l methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.

Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations. To determine the effect of fixation on the preservation of the nucleic acids, we fixed two randomly chosen fresh pathology specimens in formalin, B-5, Bouin's, Zenker's, ethanol, and Omnifix for 6, 24, 48, 72, and 168 hr (1 week), and then embedded the tissue in paraffin. Oligonucleotide primers specific for the cytoplasmic-beta-actin gene were chosen to span an intron such that amplification yielded a product of 250 BP for DNA and 154 BP for RNA. A single 6-microns section was cut from each paraffin block, deparaffinized, and then subjected to 30 rounds of amplification for either DNA or RNA. On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr of fixation time, whereas variable product was seen with formalin or Zenker's fixation; all specimens fixed in Bouin's or B-5 were negative. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr in the formalin-fixed tissue. The Zenker's-fixed tissues gave variable results, and the Bouin's and B-5 tissues gave consistent results only after 6 hr of fixation. We therefore conclude that choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material.     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and -III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraffin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.     

Resorcin Fuchsin Staining of Neurosecretory Cells

The resorcin fuchsin staining solution was prepared by dissolving 1 gm of the dry dye (Chroma) in 98 ml of 70% ethanol acidified by 2 ml of concentrated HCI. When applied to paraffin sections of vertebrate hypothalamus fixed in a modified Bouin's fluid (0.5% trichloroacetic acid replacing 5% acetic), the solution stained neurosecretory cells in a manner comparable to staining by Gomori's aldehyde fuchsin. The resorcin fuchsin solution requires no ripening and is said to keep for months. It showed no deterioration in the 20 day period of testing. Optional fixatives are: unmodified Bouin's, Heidenhain's SUSA, and alcoholic trichloroacetic acid.     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

Summary We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I,-III and-IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and-III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraftin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.     

Differentiation of Two Types of Basophils In The Adenohypophysis of The Rat and The Mouse

Pituitaries are fixed for 24 hr. in Bouin's fluid containing 0.5% trichloroacetic acid instead of 5% acetic acid, or in a mixture of 9 parts SUSA and 1 part saturated aqueous solution of picric acid. They are embedded in paraffin and horizontal sections are cut at 3-4 μ. The staining method consists of 3 phases: (a) immersion in aldehyde-fuchsin for the selective demonstration of the beta cell granules, (b) staining of the nuclei with Ehrlich's hematoxylin and (c) a rapid one-step counterstain with light green and orange G dissolved in a phosphotungstic-acetic acid mixture for the differentiation of the acidophilic and the delta cell granules.     

Differentiation of Nucleic Acids by Staining at Controlled pH and by A Schiff-Methylene Blue Sequence

Fixation with Bouin's fluid preserves cytoplasmic and nucleolar ribonucleic acid (UNA) particularly well. RNA may be demonstrated preferentially in Bouin fixed tissue by staining with 0.02% thiazine dye in aqueous McIIvaine phosphate-citrate buffer between pH 3 and 4. Methylation blockage of basophilia other than that of nucleic acids permits staining of RNA with thiazine dyes near neutrality. The deoxyribonucleic acid (DNA) of chromatin undergoes a Feulgen type hydrolysis in the tissue block during 24 hr fixation with Bouin's fluid. This hydrolysis by picric acid permits Schiff staining of the DNA wthout further acid hydrolysis. Consequently after Bouin fixation it is possible to demonstrate DNA and RNA specifically by a Schiff-methylene blue sequence. Thus a Schiff stain without further acid hydrolysis followed by 0.02% methylene blue in phosphate-citrate buffer at pH 3.0 to 3.5 colors DNA magenta in contrast to the blue of RNA.     

Pyronin Y in the Methyl-Green-Pyronin Histological Stain

Two samples of pyronin Y were found which, with the exception of eosinophilic granules and osteoid, stained only nucleic acids in animal tissues. Good differentiation was obtained. with n-butyl alcohol. It was therefore possible to prepare a differentially staining mixture of either of these pyronins combined with methyl green. This mixture stains polymerized desoxyribose nucleic acid (DNA) clear green, depolymerized DNA and ribonucleic acid red. The red staining of eosinophilic granules and osteoid is readily distinguished by its persistence after ribonuclease or warm-buffer extraction. The staining mixture consists of: (1) pyronin Y (Edward Gurr or G. T. Gurr), CHCl₃ extracted, 2% aq, 12.5 ml; (2) methyl green, CHCl₃ extracted, 2% aq, 7.5 ml; (3) distilled water, 30 ml. The staining procedure is as follows. (1) Immerse slides 6 min in the dye mixture. (2) Blot with filter paper. (3) Immerse in 2 changes of n-butyl alcohol, 5 min each. (4) Xylene, 5 min. (5) Cedar oil, 5 min. (6) Apply Permount and cover.     

THE CHEMICAL COMPOSITION OF ISOLATED CHROMOSOMES

By means of 1 M NaCl isolated lymphocyte chromosomes can be separated into two fractions, each of which contains nucleoprotein. The fraction soluble in M NaCl consists largely of desoxyribose nucleohistone, and constitutes 90 to 92 per cent of the mass of the chromosome. The insoluble residue (the residual chromosome is a coiled thread containing some 12 to 14 per cent of ribose nucleic and about one-fifth as much desoxyribose nucleic acid; the residual chromosome accounts for 8 to 10 per cent of the mass of the chromosome. The staining of chromosomes—whether by the Feulgen procedure, by hematoxylin, orcein, or by basic dyes such as crystal violet—is due to the nucleohistone fraction which contains about 96 per cent of the nucleic acid of the chromosome. The form of the chromosome is due primarily to the protein thread of the residual chromosome. This thread is the only linear structure of microscopic dimensions in the chromosome that is not readily dispersed. When chromosomes are broken, it must be supposed that a break is made in the protein thread of the residual chromosome. The foregoing provides evidence for considering the residual chromosome to be the basis of the linear order of the genes. This would mean either that the residual chromosome is a structure around which the genes are organized or that the genes form part of its substance.     

Effects of different fixatives on detection of nucleic acids from paraffin-embedded tissues by in situ hybridization using oligonucleotide probes

Detection of nucleic acids from paraffin-embedded material by in situ hybridization with oligonucleotide probes is increasingly being used. To determine the effect of fixation on the preservation of DNA and mRNA, we studied 18 lymphoid tissues fixed in B5, formalin, OmniFix, ethanol, and Bouin's fixatives and embedded in paraffin by in situ hybridization, using biotinylated oligonucleotide poly d(T) probes and immunoglobulin light chain probes. Detection of DNA using the poly d(T) probe was most consistent and most intense in tissue fixed in formalin, followed by OmniFix and ethanol, with B5 and Bouin's fixatives yielding unsatisfactory results. Detection of mRNA, using the light chain probes, was most consistent and most intense with tissue fixed in formalin and Bouin's solution, followed by B5 fixative, with OmniFix and ethanol fixatives yielding unsatisfactory results. The results of mRNA detection using the poly d(T) probe were found not to correlate with mRNA content as determined by the light chain probes for several fixatives, possibly owing to selective degradation of portions of the mRNA molecule.     

Immunocytochemical localization of nerve growth factor: effects of fixation

The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.     

Pyronin Y in the Methyl-Green-Pyronin Histological Stain

Two samples of pyronin Y were found which, with the exception of eosinophilic granules and osteoid, stained only nucleic acids in animal tissues. Good differentiation was obtained. with n-butyl alcohol. It was therefore possible to prepare a differentially staining mixture of either of these pyronins combined with methyl green. This mixture stains polymerized desoxyribose nucleic acid (DNA) clear green, depolymerized DNA and ribonucleic acid red. The red staining of eosinophilic granules and osteoid is readily distinguished by its persistence after ribonuclease or warm-buffer extraction. The staining mixture consists of: (1) pyronin Y (Edward Gurr or G. T. Gurr), CHCl₃ extracted, 2% aq, 12.5 ml; (2) methyl green, CHCl₃ extracted, 2% aq, 7.5 ml; (3) distilled water, 30 ml. The staining procedure is as follows. (1) Immerse slides 6 min in the dye mixture. (2) Blot with filter paper. (3) Immerse in 2 changes of n-butyl alcohol, 5 min each. (4) Xylene, 5 min. (5) Cedar oil, 5 min. (6) Apply Permount and cover.     

A Modified One-Step Trichrome Stain for Demonstration of Fine Connective Tissue Fibers

Gomori's one-step trichrome procedure was modified to improve coloration of fine connective tissue fibers. Paraffin sections from tissues fixed in alcohol, acetone, Zenkerformol, 10% formalin, Kaiserling's or Carnoy's fluid were mordanted 1 hr at 56 C in Bouin's solution, stained 1 min in a trichrome solution (chromotrope 2R-phosphomolybdic acidaniline blue WS) adjusted to pH 1.3 with HCl, rinsed in 1% aqueous acetic acid, dehydrated and covered. Collagen, reticulum fibers, basement membranes, ring fibers around splenic sinuses, intercalated discs in cardiac muscle and cartilage were colored blue. Nuclei, cytoplasm, fibrin, muscle fibers and elastic fibers were stained red. Pretreatment of sections with Bouin's solution enhanced the affinity of tissues for chromotrope 2R and was found essential for satisfactory coloration of material fixed in alcohol, acetone, formalin or Carnoy's fluid. Because this method does not require differentiation, it gave uniform results even in the hands of inexperienced laboratory trainees. No fading was observed in sections stored for more than 8 yr.     

A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide

We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. In contrast to the original method, the modified CTAB procedure is faster, omits the selective precipitation and CsCl gradient steps, uses less expensive and toxic reagents, requires only inexpensive laboratory equipment and is more readily adapted to high-throughput DNA extraction. This protocol yields approximately 5-30 microg of total DNA from 200 mg of tissue fresh weight, depending on plant species and tissue source. It can be completed in as little as 5-6 h.     

Immunofluorescent Labelling of K-Papovavirus Antigens in Glycol Methacrylate Embedded Material: A Method for Studying Infected Cell Populations by Fluorescence Microscopy and Histological Staining of Adjacent Sections

Trypsin and protease V (pronase) were studied for their ability to enhance immuno-fluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 μm sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.