Effects of Inorganic Oxides, Sulfides, Chlorides, and Acid Chlorides on Metachromasia and Other Staining Properties

The ability of 17 inorganic compounds (POCl₃, PSC1₃, PC1₃, P₂O₅, P₂S₅, P₄S₃, P₄S₇, PC1₅, Sb₂O₅, As₂O₅, BiOC1₂, SeOC1₂, SO₂C1₂, Sb₂S₅, VOC1₂, SiC1₄ and CrO₂Cl₂) dissolved in pyridine or 2,2,4-trimethyl pentane, to enhance subsequent staining of tissue components with toluidine blue, phosphotungstic acid-hematoxylin (PTAH), leukofuchsin, and dihydroxydinaphthyl-disulfide (DDD) was studied. Eight of these compounds were also tested for ability to enhance staining with Alcian blue 8GN and Luxol fast blue MBS. Nine of the 17 compounds produced increased staining of certain tissue components with leukofuchsin, 13 with toluidine blue, 16 with PTAH, and 16 with DDD. The results suggest additional approaches to identification of tissue entities by induced metachromatic basophilia and leukofuchsin positivity as well as by the other stains studied, and also suggest a number of hitherto unstudied modes of reaction between the dyes used and reactive groups of tissue components. Many reactions of the compounds tested, with reactive groups known to be present in tissue components, are basecatalyzed, so that choice of solvent can influence the results obtained.     

Luxol Fast Blue Mbs and Phloxine; a Stain for Mitochondria

The applicability of Luxol fast blue MBS as a 0.1% solution in 0.05% acetic acid to the staining of mitochondria, first recognized in rat kidney by Shanklin and Nassar (Stain Techn., 34: 257-60. 1959), was confirmed in various organs (formalin-Zenker and Regaud's fixations; paraffin embedding) of the mouse and bullfrog. In liver cells and in the epithelium of renal tubules, mitochondria were stained green, selectively and clearly. The dark cells of the renal tubules and the middle piece of sperms in both animals were conspicuously demonstrated by their dense assemblages of green granules. The periodic acid-Schiff procedure proposed by Shanklin and Nassar as a counterstain was replaced by staining in 0.5% aqueous phloxine, 2-3 min; differentiation in 5% phosphotungstic acid, 2 min; and washing in water, 5 min. This simplified and accelerated the techique, and gave a better color contrast. Advantages of Luxol fast blue MBS and phloxine staining over traditional methods for mitochondria in paraffin sections are: durability of the stain, high specificity, simplicity of procedure, and constant result.     

Effects of Inorganic Oxides,Sulfides, Chlorides,and Acid Chlorides on Metachromasia and Other Staining Properties

The ability of 17 inorganic compounds (POCl₃, PSC1₃, PC1₃, P₂O₅, P₂S₅, P₄S₃, P₄S₇, PC1₅, Sb₂O₅, As₂O₅, BiOC1₂, SeOC1₂, SO₂C1₂, Sb₂S₅, VOC1₂, SiC1₄ and CrO₂Cl₂) dissolved in pyridine or 2,2,4-trimethyl pentane, to enhance subsequent staining of tissue components with toluidine blue, phosphotungstic acid-hematoxylin (PTAH), leukofuchsin, and dihydroxydinaphthyl-disulfide (DDD) was studied. Eight of these compounds were also tested for ability to enhance staining with Alcian blue 8GN and Luxol fast blue MBS. Nine of the 17 compounds produced increased staining of certain tissue components with leukofuchsin, 13 with toluidine blue, 16 with PTAH, and 16 with DDD. The results suggest additional approaches to identification of tissue entities by induced metachromatic basophilia and leukofuchsin positivity as well as by the other stains studied, and also suggest a number of hitherto unstudied modes of reaction between the dyes used and reactive groups of tissue components. Many reactions of the compounds tested, with reactive groups known to be present in tissue components, are basecatalyzed, so that choice of solvent can influence the results obtained.     

Luxol Fast Blue Arn: A New Solvent Azo Dye with Improved Staining Qualities for Myelin and Phospholipids

Luxol fast blue ARN (Du Pont, C.I. solvent blue 37) is a diarylguanidine salt of a sulfonated azo dye. This dye was compared with other Luxol blue and Luxol black dyes. Luxol fast blue ARN has improved staining qualities for phospholipids and myelin, and can advantageously be substituted for Luxol fast blue MBS (MBSN). Appropriate staining times for a 0.1% dye solution in 95% ethanol (containing 0.02% acetic add) at 35°-40° C range from 2-3 hr. After staining, the sections should be rinsed in 95% ethanol, rinsed in distilled water, and differentiated for 2 sec in 0.005% Li₂CO₃, rinsed in 70% ethanol, washed in water, and counterstained as required. Phospholipids and myelin selectively stain deep blue. A fixative containing CaCl₂, 1%; cetyltrimethylammonium bromide, 0.5%; and formaldehyde, 10%, in water gave excellent results with brain. However, 10% formalin can be used. The staining of the phospholipids is probably due to the formation of dye-phospholipid complexes.     

Preparation of Large Epoxy Sections for Light Microscopy as an Adjunct to Fine-Structure Studies

Tissue blocks 2 × 2 × 0.4 cm were fixed 6-24 hr in phosphate-buffered 5% glutaraldehyde then sliced to 2 × 2 × 0.1 cm and soaked in 0.1 phosphate-buffer (pH 7.3) for at least 12 hr. Fixation was continued for 2 hr in phosphate-buffered 1-2% OsO₄. The slices were dehydrated, infiltrated with Araldite, and embedded in flat-bottomed plastic molds. Sectioning at 1-8 μ with a sliding microtome was facilitated by addition of 10% dibutylphthalate to the standard epoxy mixture. The sections were spread on warm 1% gelatin and attached to glass slides by drying, baking at 60 C, fixing in 10% formalin or 5% glutaraldehyde and baking again. Sections were mordanted in 5% KMnO₄ (5 min), bleached with 5% oxalic acid (5 min) and neutralized in 1% Li₂CO₃ (1 min). Several stains could then be applied: azure B, toluidine blue, azure B-malachite green, Stirling's gentian violet, MacCallum's stain (modified), tribasic stain (modified) and phosphotungstic acid-hematoxylin. Nuclei, mitochondria, specific granules, elastic tissue or collagen were selectively emphasized by appropriate choice of staining procedures, and cytologic detail in 1-3 μ sections was superior to that shown by conventional methods. Selected areas from adjacent 4-8 μ sections could be re-embedded for ultramicrotomy and electron microscopy.     

Staining Mitochondria in Saccharomyces cerevisiae

After testing various procedures (amidoblack 10B, acid fuchsin-methyl blue, Luxol fast blue MBS-phloxine, toluidine blue O, Jams green B and pinacyanol), three stains can be recommended for staining both types of mitochondria (globose and threadlike) in the cells of Saccharomyces cerevisiae: (1) 0.1% solution of amidoblack 10B in citrate buffer (pH 3.0) for 10 min; (2) 0.01% solution of toluidine blue O in phosphate buffer (pH 6.0) for 30 min; (3) 0.01% solution of Janus green B in distilled water (pH 5.6) for 30 min. The latter stain is most specific because its staining reaction depends upon the action of the mitochondrial enzyme cytochrome c oxidase. Yet, low concentrations and short incubation periods must be applied to avoid poisoning of the cell metabolism.     

Fixation and Staining of Planaria for Histological Study

Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acld-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH.     

Luxol Fast Blue as a Stain for Mallory's Alcoholic Hyaline

Luxol fast blue MBS (du Pont), which has frequently been used as a stain for phospholipids, stains Mallory's “alcoholic” hyaline a deep purplish blue. The stain is stable and provides histological appearances far superior to other methods. It is used on paraffin sections of tissue fixed in formalin or formalin-sublimate as a 0.1% solution in 90% alcohol at 60°C for 8 hr. Differentiation is made with 0.05% Li₂CO₃ and a red counterstain applied.     

Acceleration of Mallory's Phosphotungstic Acid-Hematoxylin Staining for Skeletal Muscle Fixed in Formalin

The staining time for mammalian skeletal muscle fixed in neutral phosphate-buffered formalin was shortened from 12-24 hr to 10-30 min. The permanganate-oxalate sequence was omitted although oxidation by periodic acid or with iodine was found to be necessary. The material was embedded in paraffin and cut 6 μ or less. Deparaffinized sections were treated with 1% alcoholic iodine for 10 rain followed by 5% Na₂S₂O₃ for 2 min and placed in an oven at 60 C for 10-30 min to stain in a preheated mixture of 50 ml of ripened Mallory's phosphotungstic acid-hematoxylin and 1 ml of 2% phosphomolybdic acid. Experiments with fixation showed that the staining procedure followed Zenker's fluid successfully but not Bouin's fluid. Oxidation by KMnO₄ was effective only after Zenker fixation; oxidation by CrO₃ was unsuccessful.     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.     

Displacement

Displacement is a noncommital term for the reactions that occur when slides previously stained in phloxine or rose Bengal are immersed for varying lengths of time in a solution of another dye in ethyl Cellosolve. In most histotechnic tests Lendrum's (1947) phloxine-tartrazine is given as the stain for acidophilic inclusion bodies. However the lack of contrast between the phloxine and tartrazine has been a serious limitation. A number of dyes were tried as possible substitutes for the tartrazine. A rose Bengal-Bismark brown Y procedure was developed which stains similarly to Lendrum's phloxine-tartrazine and which doer have the needed contrast. After staining for 10 min in 1% aqueous rose Bengal and rinsing in isopropyl alcohol slides are placed for 20, 30, 40 and 50 min in 0.05% Bismark brown Y in ethyl Cellosolve. In various tissues and structures the rose Bengal is sequentially displaced by the Bismark brown Y. Thus collagen loses the red stain after 30 min while acidophilic structures like sperm heads and Paneth cell granules retain the red stain after 50 min in the displacement solution. The results are strikingly similar to staining with alkaline Biebrich scarlet.     

Luxol Fast Blue Combined With the Periodic Acid-Schiff Procedure for Cytological Staining of Kidney

Rat kidneys fixed in Regaud's fluid were stained by luxol fast blue (LFB), by the periodic acid-Schiff (PAS) method, and by LFB combined with PAS. When used separately the PAS stains the brush border, hyaline droplets and basement membranes reddish, the LFB stains the mitochondria, hyaline bodies and basement membranes greenish-blue. The combined LFB-PAS method stains the brush border reddish and the mitochondria dark blue, while the hyaline bodies and basement membranes are purplish colored. The LFB-PAS method provides color contrasts which show cytological features that are particularly significant in the kidney.     

Displacement.

Displacement is a noncommital term for the reactions that occur when slides previously stained in phloxine or rose Bengal are immersed for varying lengths of time in a solution of another dye in ethyl Cellosolve. In most histotechnic texts Lendrum's (1947) phloxine-tartrazine is given as the stain for acidophilic inclusion bodies. However the lack between the phloxine and tartrazine has been a serious limitation. A number of dyes were tried as possible substitutes for the tartrazine. A rose Bengal-Bismark brown Y procedure was developed which stains similarly to Lendrum's phloxine-tartrazine and which does have the needed contrast. After staining for 10 min in 1% aqueous rose Bengal and rinsing in isopropyl alcohol slides are placed for 20, 30, 40 and 50 min in 0.05% Bismark brown Y in ethyl Cellosolve. In various tissues and structures the rose Bengal is sequentially displaced by the Bismark brown Y. Thus collagen loses the red stain after 30 min while acedophilic structures like sperm heads and Paneth cell granules retain the red stain after 50 min in the displacement solution. The results are strikingly similar to staining with alkaline Biebrich scarlet.     

Studies on Textile Dyes for Biological Staining II. A Trichrome Stain for General Use

This trichrome staining procedure differentially stains elastic fibers, collagen fibers and mucin. Gomori's aldehyde-fuchsin is used for elastic fibers; fast yellow TN is the component used for collagen and cytoplasm; pontacyl blue black SX is the nuclear stain. Procedure: Paraffin sections to water; aldehyde-fuchsin, 30 min; 70% ethanol; distilled water; 0.75% pontacyl blue black SX in 1.5% K.₂Cr₂O₇, 15 min; tap water; 70% ethanol to wash off all free dye; 2% fast yellow TN in 95% ethanol, 5 min; dehydrate, clear and cover.     

Luxol Fast Blue as a Selective Stain for Alpha Cells in the Human Pituitary

Human pituitaries fixed in Bouin's fluid or 10% formalin were stained by the PAS, Masson trichrome and luxol fast blue methods. By comparing adjacent sections stained by these 3 methods it was found that the alpha cells which are PAS negative, but stained red by the Masson trichrome method, were intensely stained by luxol fast blue. The beta cells which are stained blue by the PAS and Masson methods were not stained by luxol fast blue. Similar observations were made on a series of pituitaries from 8 other mammalian species. It is concluded that luxol fast blue is a selective stain for alpha cells in the mammalian pituitary.     

A Simple Toluidine Blue—Basic Fuchsin Stain for Spermatozoa in Epoxy Sections

Differential staining of cell components of spermatozoa is readily accomplished in Epon or Araldite sections 0.5-1 μ thick from rat and hamster testis and epididymis, and stained as follows: 1% aqueous toluidine blue buffered at pH 6, 0.5-3 min at 90 C; washed in distilled water; 1% basic fuchsin in 50% alcohol, 3-5 min at 20-25 C; differentiated with 70% alcohol; allowed to dry; and mounted in a resin of high refraction (DPX was used). Results: acrosome, bright magenta; nucleus, deep blue; mitochondrial sheath of the middle-piece, pinkish purple; and tail, pale red. This procedure combined with staining of collagen by applying 2% aqueous phosphotungstic acid 1-2 min as a mordant, followed by 1% light green in 50% alcohol containing 1% acetic acid, 1-2 min at 20-25 C, gives polychromatic staining and is useful as a general stain for other epoxy-embedded tissues.     

A New Principle in Polychrome Staining: A System of Automated Staining, Complementary to Hematoxylin and Eosin, and Usable as a Research Tool

A staining system is described in which each stage forms a separate module or unit. All reagents, concentrations of dye, ratios of phosphotungstic acid to dye, pH values, temperature and staining times are standardized and only aqueous solutions used. The technic uses equal strength solutions of orange G, acid fuchsin and methyl (or aniline) blue, in ascending order of molecular size, at pH 2.5 (range: 2.3 to 2.7). Phosphotungstic acid is incorporated in the dyebaths, not used separately, and the combination of this with ferric alum hematoxylin (Lillie's by preference) and either naphthol yellow S or picric acid as a primer, enables fibrin and cytoplasmic components to be demonstrated vividly, with other tissues shown in clear contrasting colors. Erythrocytes are yellow, fibrin red and collagen blue. The system permits substitution of dyes, lending itself to both manual and computer recording and analysis, helped by a notation system for identifying variants. Many of the factors are variable at will. The system aids research into the mechanism of polychrome staining, and, by extrapolation, into the mechanism of action of other stains. Two manually or machine usable progressive polychrome technics intended for routine use are described. They identify tissue components consistently, complementing the standard hematoxylin and eosin stain, and deserve equal attention during reporting. Variants may be used for one-minute one-stage staining of frozen sections, or to give strong colors with 2 mμ acrylic sections.     

Epoxy Embedding, Sectioning and Staining of Plant Material for Light Microscopy

By using a formula which gives a relatively soft epoxy embedding medium, it is possible to cut sections of plant material with a sliding microtome equipped with a regular steel knife. Blocks having a cutting face of 10 × 10 mm, giving sections of 4-10 μm, can be used. Tissues are fixed in Karnovsky's fluid, postfixed in 1 or 2% OsO₄, embedded in Spurr's soft epoxy resin, Araldite, or Epon mixtures. 5% KMnO₄, followed by 5% oxalic acid, then neutralized in 1% LiCO₃, are used to mordant the sections. Some of the stains used are Mallory's phosphotungstic acid-hemotoxylin, acid fuchsin and toluidine blue, or toluidine blue. Mounting is done with whichever soft epoxy resin was used in casting the blocks.     

Arginine-rich proteins in spherical inclusions of human locus coeruleus neurons demonstrated by benzil modification

Previous studies have shown that aminergic neurons in the normal human brain contain acidophilic cytoplasmic inclusions--called protein bodies (PBs)--that are reduced or absent in parkinsonism and disrupted in depression. The purpose of the present study was to elucidate the constitution of PBs in five formalin-fixed normal human brains using histochemical methods specific for histones, protamines, and the amino acid arginine. PBs were revealed with alkaline fast green and bromphenol blue, exhibiting a high content in histones and in protamines. They developed blue metachromasia with phosphotungstic acid-hematoxylin and green fluorescence with phenanthrenequinone, which established the presence of arginyl residues. Using benzil, which selectively modifies the guanido group of arginine, staining was blocked for each of the above two methods. The application of Mallory's trichrome procedure after benzil differentiated the PBs into an unstained core and a still fuchsinophilic rim. Since the fuchsinophilia of the rim was shown to persist after acetylation as well, we suggest that this rim probably contains acidic macromolecules that attach to the basic charges of the amphoteric acid fuchsin. We conclude that the PB are complex structures consisting of a core segregating arginine-rich proteins and a rim which probably contains macromolecules of an acidic nature.