Double-enzyme conjugates, producing an intermediate color, for simultaneous and direct detection of three different intracellular immunoglobulin determinants with only two enzymes

A new double-enzyme conjugate was synthesized by coupling alkaline phosphatase (AP) to horseradish peroxidase (HRP). After AP (blue) and subsequent HRP (red) cytochemistry, this new conjugate produced a stable intermediate-colored (violet) product. By coupling this double-enzyme conjugate to an antigen (trinitrophenyl, TNP) or an antibody (anti-mouse immunoglobulin G2a), anti-TNP or -IgG2a-producing cells could be demonstrated as violet cells in spleen sections. This led to the development of a rapid one-step incubation--two-step cytochemical procedure for simultaneous detection of three different determinants in a single tissue section. To demonstrate this novel triple staining method, we coupled three different antigens to, respectively, AP, HRP, and AP-HRP. When spleen sections of immunized animals were incubated with a mixture of these three antigen-enzyme conjugates, we could distinguish antibody-forming cells against each of these three antigens simultaneously as red (HRP), blue (AP), and violet (AP-HRP) cells. The simultaneous detection of three different classes of intracellular antibodies in a single section also proved to be possible with this method. With this study we provide a new direct method for detection of three different intracellular immunoglobulins after a one-step incubation and a two-step standard cytochemical procedure.     

Understanding microwave-stimulated Romanowsky-Giemsa staining of plastic embedded bone marrow

Summary Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky-Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky-Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.     

A Mixture of Methyl Green and Pyronin for Cell Fractionation Studies

A staining mixture consisting of 0.57% methyl green and 0.1 1 % pyronin B (calculated from the actual dye content) dis- solved in glycerol, 20 ml.; 2% aqueous phenol, 100 ml.; and 95% ethanol, 25 ml., was found to be optimum for differentiating cell components containing desoxypentose and pentose nucleic acids. The stain can be used for either fresh suspensions or unfixed dried smears of tissue homogenates. Nuclei are stained bright blue, and nucleoli and cytoplasmic particles, bright pink.     

Neuron Staining by Basic Dyes Versus Basic Metal-Dye Complexes; Differences Shown by Histochemical Blocking Reactions

Various blocking procedures were applied to sections of paraffin-embedded, formalin-fixed cat spinal cord. Treated sections and untreated controls were stained with cresyl violet acetate or gallocyanine-chrome alum. Although both dyes have been said to stain by simple salt formation it was found that staining was affected differently for each dye by the blocking procedures, and also that staining of neuron nuclei differed in the controls. In these, the cresyl violet acetate stained only the nucleoli within the nucleoplasm whereas gallocyanine-chrome alum stained much more material of unknown composition and function. It is proposed that if cresyl violet acetate and other basic dyes stain by salt linkage, and can be specific for nucleic acid and other highly acid materials, then gallocyanine and other basic metal dye complexes can not be specific for nucleic acid and do not stain by a simple salt linkage.     

Techniques for Studying Adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are frozen rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl₂ (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl₂ (2.5%). When procedures described above for immature adipose depots are combined with esterase fining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

Techniques for studying adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl2 (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl2 (2.5%). When procedures described above for immature adipose depots are combined with esterase staining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

Contribution to the Staining of Neuroglia

The chemistry of Weigert's glia staining method is critically discussed. An investigation of the Heidelberger Victoria blue staining method has shown that Victoria blue may be replaced by other phenylmethane dyes as methyl violet, ethyl violet, and crystal violet. It was found that the exposure of the stained section to sunlight is an oxidation process. Artificial ultra violet rays or chemical oxidation agents give the same effect. Frozen sections fixed in formalin or alcohol may be stained in a concentrated aqueous solution of any of the above mentioned phenylmethane dyes, dried, and exposed to ultra violet rays for 30 minutes, then treated with 1/10 N. iodine solution, differentiated in xylol anilin and cleared in xylol. The glia cell body as well as the fibrils are clearly differentiated from the nervous elements and connective tissue.     

The Germicidal Effect of Staining Solutions

Gentian violet, crystal violet and carbol fuchsin applied to cover slip preparations for one minute will destroy the majority of non-spore-forming bacteria and yeasts, tho they can not be relied upon to do this consistently and in all cases.

The Gram staining procedure is more effective and non-spore-formers were never found to survive this process.

Methylene blue stains exert very little if any germicidal power and most organisms survived them readily. India ink was totally ineffective.

Several species of yeasts and yeast-like molds were killed in every instance by the Gram stain, gentian violet, crystal violet and carbol fuchsin, but survived both Loeffler's methylene blue and a plain aqueous solution of methylene blue.     

The Germicidal Effect of Staining Solutions

Gentian violet, crystal violet and carbol fuchsin applied to cover slip preparations for one minute will destroy the majority of non-spore-forming bacteria and yeasts, tho they can not be relied upon to do this consistently and in all cases.

The Gram staining procedure is more effective and non-spore-formers were never found to survive this process.

Methylene blue stains exert very little if any germicidal power and most organisms survived them readily. India ink was totally ineffective.

Several species of yeasts and yeast-like molds were killed in every instance by the Gram stain, gentian violet, crystal violet and carbol fuchsin, but survived both Loeffler's methylene blue and a plain aqueous solution of methylene blue.     

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.     

Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining

Background

For decades, the Vibratome served as a standard laboratory resource for sectioning fresh and fixed tissues. In skilled hands, high quality and consistent fresh unfixed tissue sections can be produced using a Vibratome but the sectioning procedure is extremely time consuming. In this study, we conducted a systematic comparison between the Vibratome and a new approach to section fresh unfixed tissues using a Compresstome. We used a Vibratome and a Compresstome to cut fresh unfixed lymphoid and genital non-human primate tissues then used in situ tetramer staining to label virus-specific CD8 T cells and immunofluorescent counter-staining to label B and T cells. We compared the Vibratome and Compresstome in five different sectioning parameters: speed of cutting, chilling capability, specimen stabilization, size of section, and section/staining quality.

Results

Overall, the Compresstome and Vibratome both produced high quality sections from unfixed spleen, lymph node, vagina, cervix, and uterus, and subsequent immunofluorescent staining was equivalent. The Compresstome however, offered distinct advantages; producing sections approximately 5 times faster than the Vibratome, cutting tissue sections more easily, and allowing production of larger sections.

Conclusions

A Compresstome can be used to generate fresh unfixed primate lymph node, spleen, vagina, cervix and uterus sections, and is superior to a Vibratome in cutting these fresh tissues.     

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.     

Cytochemical differentiation of nucleic acids with a Schiff-methylene blue sequence.

A method is described whereby a Feulgen type of hydrolysis of deoxyribonucleic acid is carried out on paraffin sections of routinely fixed tissues by controlled exposure of the sections to Bouin's fluid. Subsequent staining with Schiff reagent followed by methylene blue distinguishes red-to purple-stained deoxyribonucleic acid from blue-stained ribonucleic acid. This Schiff-methylene blue sequence visualizes ribonucleic acid in nucleoli and the chromidial substance of various normal and neoplastic cells and provides an assessment of their protein synthetic activity. The method has proved valuable in demonstrating normal immunocytes and immunoglobulin-forming tumor cells in pathologic specimens.     

A novel technique for immunohistoperoxidase staining of unfixed whole joints of small animals

Summary A method has been developed to cut unfixed and undecalcified sections of rat paws from animals with adjuvant arthritis and to stain them by a biotin-avidin immunoperoxidase technique. Good tissue integrity and morphology throughout the immunohistochemical procedure were retained if the sections were first mounted on transparent sellotape. The method is illustrated with two monoclonal antibodies (mAb) and is generally applicable with any mAb or polyclonal antibody and with joints from other small animals. For rats with adjuvant arthritis it was found important to block endogenous peroxidase before immunostaining. Complete inhibition of this enzyme without loss of antigenicity was best achieved after application of the mAb and biotinylated anti-IgG conjugate to the unfixed tissue sections.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry.

In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using 35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using 35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (less than 1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Maxilon Blue Rl: A New Metachromatic Monoazo Dye

Maxilon blue RL, a basic monoazo dye developed by Geigy S. A., stains metachromatically acid mucopolysaccharide-containing elements in histological sections. This property is due to a chromatographically pure blue fraction which is the main component of the dye. The following routine has been developed for staining sections of formalin-calcium fixed paraffin-embedded tissues. Dewax in xylene and hydrate the sections through alcohol; stain in 0.05% aqueous Maxilon blue RL, from 30-60 sec; wash in distilled water; dehydrate either in tertiary butanol, 2 to 3 min, after removing excess water from the slide by blotting; or, rinse in 70% ethyl alcohol and dehydrate in 2 changes, 2 min each, of absolute alcohol; clear in xylene; mount in DPX or in Canada balsam. Acid mucopolysaccharides are colored red to violet; other basophilic elements, blue.     

Gram staining and lectin binding properties of Myxosporea and Sporozoea

The staining method developed by Christian Gram was introduced as a simple and highly selective tool for demonstrating myxosporean and coccidian sporogonic stages. When using standard blood staining procedures for those enigmatic parasites it is sometimes difficult to distinguish them from fish host tissue. They clearly exhibit a partial Gram-positive reaction in histological sections, but staining is variable in air dried fish organ imprints. To visualize the Gram-negative background of different host tissue components in histological sections, the conventional safranin counterstain of the Gram protocol may be modified as follows: after application of 2% crystal violet (basic violet 3) and Lugol's solution, sections are stained with 0.1% nuclear fast red-5% aluminum sulfate and 0.35% aniline blue (acid blue 22) dissolved in saturated aqueous picric acid. Replacement of the Gram-specific dye crystal violet with 2% malachite green gave similar results in organ imprints containing myxospores or coccidia, but only in sections containing myxosporea. Staining for 1 min with an aqueous solution of 0.5% malachite green and followed 1 min washing was sufficient for rapidly demonstrating the parasite spores in organ imprints of both myxosores and oocysts. With regard to the role of acid mucopolysaccharides and other carbohydrates in the Gram reaction of spores, alcian blue 8GX staining was compared to the binding of FITC-labeled WGA, GS I and GS II. Each lectin was applied at 20 μl/ml PBS, HEPES for 1 hr. Whereas WGA yielded a nonspecific pattern like the alcian blue staining, GS II resulted in a pattern similar to the Gram staining results. This binding was weak in untreated specimens, but was significantly enhanced when digested first within trypsin overnight in a humid chamber at 37 °C. The binding of GS II to both myxosporidian and coccidian spores suggests that they are both composed of polymers containing N-acetyl-D-glucosamine residues. Furthermore, the results suggest that this hexosamine plays a key role in the Gram reaction.     

The Gram Staining Mechanism of Cat Tongue Keratin. II. The Role of Potassium Iodide-Iodine Solution

The aniline-xylene decolorizer of the Gram-Weigert staining procedure failed to remove crystal violet dye from stained sections of cat tongue without prior treatment of the sections with potassium iodide-iodine solution. The potassium iodide of the iodide-iodine solution was found to release the major part of the crystal violet dye bound by the tongue sections. Iodine appeared also to play a role in dye release, but only to a slight degree. The amount of Gram-positive staining was increased both by alkaline treatment of the tissue prior to staining, and by increasing the pH of the iodide-iodine solution.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Summary In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using³⁵S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using³⁵S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Basic Fuchsin-Crystal Violet; A Rapid Staining Sequence for Juxtaglomerular Granular Cells Embedded in Epoxy Resin

A basic fuchsin-crystal violet staining sequence for demonstration of juxtaglomerular granular cells in epoxy-embedded tissues is rapid and results in slides with excellent contrast and intensity. Procedure: Cut sections 0.3-0.6 μ thick. Hydrate through xylene and alcohol to water. Stain in modified Goodpasture's stain (basic fuchsin, 1; aniline, 1; phenol, 1; 30% alcohol, 100) for 20-30 sec; rinse in tap water; stain in modified Stirling's (crystal violet, 5; alcohol, 10; aniline, 2; water, 88) for 20-30 sec; rinse in tap water and dry on a hotplate; mount in a synthetic resin. Granular cells of the juxtaglomerular apparatus are stained an intense dark blue by the crystal violet. Arterial elastic membranes and collagen are pale blue. Other structures are shades of red.