An Improved Method of Toluidine Blue Counterstaining of Neurons in Immunocytochemical Preparations

Immunocytochemical methods can identify individual neurons and processes made immunoreactive by virtue of the antigens they contain. However, frequently it is also useful to visualize surrounding nonimmunoreactive cells, but immunocytochemical procedures often interfere with the quality of subsequent counterstaining. This report describes an improved method of counterstaining immunocytochemical specimens with either aged (at least 1 year) or concentrated solutions of toluidine blue. This technique combines well with immunocytochemical preparations of at least two antigens, i.e., choline acetyltransferase and glutamic acid decarboxylase, to delineate nonimmunoreactive somata. Additionally, a method of photographing these color preparations is described that, by the use of an appropriate filter, allows one to illustrate sections essentially with and without blue counterstain in black and white photomicrographs.     

Improved indirect fluorescence immunocytochemical method using counterstains.

Immunocytochemistry in recent years has provided powerful tools for research in neurobiology. One of the more popular techniques is the indirect fluorescence technique in which fluorescein isothiocyanate (FITC) or tetrahodamine isothiocyanate (TRITC) is used. Although widely used, this technique has two disadvantages: (1) localization may be difficult in relation to background morphology, and (2) the fluorescence fades. The study reported here describes a modification of an indirect immunocytochemical technique using FITC, TRITC and 7-amino-4-methyl-coumarin-3-acetic acid (AMCA) which enhances localization and significantly prolongs fluorescence. Evans blue was used as a counterstain. The results show that FITC and AMCA stained cells are seen against a background of clearly distinguishable cells after counterstaining with Evans blue. However, Evans blue is not compatible with TRITC. In addition, the fluorescence life of the FITC is extended from several days to several weeks with Evans blue. These results clearly indicate that Evans blue can be used to improve indirect fluorescence immunocytochemical techniques.     

The use of trypan blue for counterstaining in the Sepharose bead immunofluorescence test. The application of the test for the demonstration of primate retrovirus-specific antibodies and antigens.

The Sepharose bead immunoflurorescence test was performed by counterstaining the beads with trypan blue. This results in a red staining of negative beads which allows an easy distinction from positive green-fluorescent beads. Sepharose beads conjugated with viral proteins or antiviral antibodies were used to demonstrate Mason-Pfizer monkey virus (MPMW)- and simian sarcoma virus (SSV) - specific antigens or antibodies. The test shows a high sensitivity and specificity and needs a small amount of material.     

Application of the avidin-biotin-complex method for the light microscopic analysis of lymphocyte subsets with monoclonal antibodies on air-dried smears

A study was undertaken of the application of the avidin-biotin-peroxidase complex (ABC) method to the monoclonal antibody MAbs staining of mononuclear cells in hematologic and cytodiagnostic materials. Satisfactory cell morphology and immunoreactivity of surface antigens were observed when the slides were fixed in 80% acetone in phosphate-buffered saline or in 60% acetone in 0.03 M citric acid buffer solution (pH 5.4). Unstained air-dried preparations could be preserved for two weeks at room temperature in a desiccator and for one year at -70 degrees C after fixation. An excellent immunoreaction, even with a weak surface antigen, was observed by inhibition of endogenous peroxidase after the secondary antibody reaction; reactions of weak antigens tended to be obscured when the inhibition was performed before the first antibody reaction. Use of the Giemsa stain as a counterstain made it possible to readily observe the cell morphology; therefore, white blood cell analysis could be performed simultaneously when peripheral blood smears were studied. The positive rate of immunoreaction by an immunofluorescent method was well correlated with that obtained by the ABC method. The ABC method proved to be an excellent immunocytochemical technique for detecting cell surface antigens with high sensitivity and specificity; furthermore, it is useful for cell morphology studies and yields permanent preparations.     

Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin and enzyme immunocytochemistry of target neurons

By the neuroanatomical tracing technique based on uptake, transport, and immunocytochemical detection of injected Phaseolus vulgaris leucoagglutinin (PHA-L), fiber trajectories of labeled neurons can be followed with great accuracy to their termination areas. To further analyze the connectivity of these fibers, the target neurons must be chemically characterized. In vibratome and frozen sections of rat brain, we tried to visualize PHA-L-labeled fibers and, simultaneously, the target neuron-related antigen. As a model system we used the projection from the pre-frontal cortex to histaminergic neurons in the posterior hypothalamic region. We tested "sequential" and "pooled" immunocytochemical procedures. In the sequential procedure, the two antigens are detected by two successive and complete immunocytochemical staining procedures, with primary antibodies raised in different animal species and with different chromogens for the final visualization. In the pooled procedure, the sections are incubated with mixtures of primary and secondary antibodies, after which the procedure is similar to the sequential procedure. We obtained excellent results on vibratome sections with a sequential procedure using first conventional peroxidase immunocytochemistry (goat anti-PHA-L primary antibody) to visualize the transported PHA-L (brown reaction product), and subsequently alkaline phosphatase immunocytochemistry (rabbit anti-histidine decarboxylase primary antibody) to locate the histaminergic neurons (blue reaction product). The resulting preparations deteriorate, however, after 1-2 months of storage. Good results were also obtained with a double peroxidase procedure on frozen sections, using nickel-enhanced diaminobenzidine to visualize the PHA-L (dark blue reaction product), and diaminobenzidine (brown reaction product) to visualize the second antigen. The quality of these preparations is permanent.     

Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations

A technique for performing correlated light and electron microscopic immunocytochemical studies on whole mount preparations has been developed using myenteric plexus from guinea pig small intestine as a model. With this method a structure containing a particular antigen can first be located by light microscopy and then examined with the electron microscope. Pieces of intestine pinned on balsa were incubated in oxygenated Krebs solution at 37 degrees C for 90-120 min and then fixed for 1 hr at room temperature in 4% formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4. The tissue was washed vigorously in several changes of 50% ethanol until the picric acid had been removed, stored overnight in phosphate buffer, and then exposed to 0.1% sodium cyanoborohydride in buffer for 30 min. Vasoactive intestinal peptide (VIP) was localized in separated layers containing myenteric plexus and longitudinal muscle using the peroxidase-antiperoxidase technique with imidazole intensification of the diaminobenzidine reaction product. At the light microscope level, tissue stained by this technique showed VIP-immunoreactive nerve cell bodies and processes throughout the thickness of the myenteric ganglia in numbers approximately equivalent to those seen in whole mounts processed by an established technique for the light microscopic demonstration of VIP, which does not involve exposure of tissue to glutaraldehyde. VIP-immunoreactive structures that were first identified at the light microscope level were subsequently examined at the electron microscope level. VIP-immunoreactive axon profiles were found to form synapses on both immunoreactive and nonimmunoreactive myenteric neurons. The fine structural appearance of the different cell types present in whole mount preparations prepared by this method was similar to that seen in conventionally fixed tissue, except that free and bound ribosomes were absent from the tissue processed for immunocytochemistry. The method described here is reliable and no more difficult than presently available methods for preembedding electron microscopic immunocytochemistry on sections. Its main advantage is that immunoreactive structures for ultrastructural study can be selected from the entire population of chemically identified nerves within a whole mount rather than from a smaller sample present within a section. This technique is applicable to other tissues that can be stained immunohistochemically in whole mounts. The fixation and penetration enhancement procedures can also be adapted for immunocytochemical studies on vibratome or frozen sections.     

Maillet's Oso4-ZnI2 Fixative and Alcian Blue Staining in the Study of Neurosecretion; Invertebrates

Maillet's OsO₄-ZnI₂ fixation staining can be combined with a subsequent counterstaining by Alcian blue or aldehyde fuchsin to demonstrate neurosecretory cells in addition to cytological details of the nerve tissue. This technic has been applied to various annelids: Eisenia foetida (Oligochaeta), Erpobdella octoculata (Achaeta) and Nereis diversicolor (Polychaeta). The material is fixed in a 1:4 mixture of 2% OsO₄ and 3% ZnI₂ for 15 nr, embedded in paraffin, sectioned at 5 μ and the sections alternatively mounted on two glass slides. One of these is oxidized by a solution of 0.3% KMnO₄ acidified by 0.6% H₂SO₄ and counterstained with 1% Alcian blue, pH 0.2, the other one is mounted in balsam. The two preparations may then be compared to locate the neurosecretory cells among the other neurons shown on a slide treated only by the OsO₄-ZnI₂. Secretory cells are not stained by Maillet's reagent; except for their Golgi bodies and their cellular and nuclear membranes. The zone of grains which is generally strongly stained by the Alcian blue takes a yellowish hue from the OsO₄-ZnI₂ fixation. This method could be successfully applied to the histological controls in regeneration experiments. In these last ones, we must simultaneously observe the regeneration of the nervous fibres and the possibility of intervention of neurosecretory elements.     

Preparation, Purification, and Stability of Tuberculin

The method used to produce “Connaught” tuberculin purified protein derivative (PPD) is described. The tuberculin PPD for the multiple-puncture method was shown to be stable for at least 24 months at 5 C; tuberculin PPD for the intracutaneous method was shown to be stable at 5 C and 24 C for a period of 18 months in the presence of Tween 80. Evans blue or brillant vital red was added to tuberculin PPD for improved testing by the multiple-puncture method. These tinted tuberculin preparations were found to be as stable as the Connaught tuberculin PPD preparations without dye at 5 C. Freeze-dried tuberculin PPD with Plasdone as an inert base was found to be remarkably stable for a period of at least 24 months at 5, 24, and 37 C.     

The dye-exclusion test for cell viability: Persistence of differential staining following fixation

Summary A method is described whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differneces that are retained following aldehyde fixation. This work was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada.     

Use of immunoperoxidase and immunogold methods in studying prolactin secretion and application of immunogold labelling for pituitary hormones and neuropeptides

Two immunocytochemical methods, immunoperoxidase and immunogold (IG), were used in an attempt to study the dynamic process of prolactin release from stimulated rat pituitary mammotrophs. The immunogold method was also used to localize other pituitary hormones including growth hormone, follicle-stimulating hormone, luteinizing hormone, and the neuropeptides substance P, neuropeptide tyrosine, leu-enkephalin, and atrial natriuretic factor in peripheral nerves. Light-microscopic immunoperoxidase staining of prolactin revealed a unique distribution of immunoreactive mammotrophs. Two groups of cells were seen, one centrally located and one forming a narrow peripheral rim on the gland. The two groups were separated by a zone of nonimmunoreactive cells. In addition, the distribution of immunoperoxidase-stained material was not uniform in all mammotrophs. In some, prolactin immunoreactive material was clumped near the nucleus (in the Golgi cisternae); in others it was more diffused within the cytoplasm (but immediately surrounding the cisternae of rough endoplasmic reticulum). After stimulation of mammotrophs, via suckling, prolactin-immunoreactive material was visualized in extracellular spaces. With immunogold methods, prolactin labelling was seen mainly in secretory granules; but some labelling of Golgi cisternae and rough endoplasmic reticulum also occurred. Immunogold labelling revealed that material immunoreactive for leu-enkephalin and atrial natriuretic factor was present in nerve terminals in the rat paracervical ganglion. Material immunoreactive for substance P and neuropeptide tyrosine was present in nerve terminals in the guinea pig heart. Thus, in some situations the immunoperoxidase technique was useful and helped to visualize "grossly" the presence of specific antigens, but it was inadequate for fine ultrastructural localization of these antigens. The immunogold technique was excellent for precise localization of antigens and especially for the detection of colocalization of different antigens. This method can be used in very different structures, such as the adenohypophysis and peripheral nervous tissue, without any modification except for the nature of the antibodies.     

The dye-exclusion test for cell viability: Persistence of differential staining following fixation

Summary A method is described whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differences that are retained following aldehyde fixation. This work was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada. Recipient of a Research Studentship from the National Cancer Institute of Canada.     

Estimation of Cytotoxic Injury to Gastric Parietal Cells by Trypan Blue Exclusion Test Followed by Hematoxylin-Eosin Counterstaining of Fixed Smears

A modification of a trypan blue exclusion technique for detection of cyto-toxicity is described with the use of a trypan blue exclusion test followed in sequence by hematoxylin-eosin counterstaining of smears permanently fixed. This technique, as applied to the suspension of parietal cells, permits the distinction between intact, moderately damaged, and severely damaged cells, determination of their ratio to each other, and an insight into the interrelations of aggressive cells (sensitized lymphocytes) to target cells (parietal cells) in the permanently fixed gastric cells suspension.     

Double immunoenzymatic staining for the simultaneous detection of Epstein-Barr virus induced antigens

A double indirect immunoenzymatic staining was developed for the simultaneous visualization of Epstein-Barr virus-induced early antigens and virus capsid antigens in P3HR1 lymphoblastoid cell line. The double immunocytochemical staining was performed with a four-stage and a two-stage procedure employing human sera and monoclonal antibodies against Epstein-Barr virus-induced antigens, followed by the addition of specific alkaline phosphatase and peroxidase labeled antisera. The selection of substrates yielding reaction products of contrasting colours enabled the observer to distinguish cells expressing Epstein-Barr virus capsid antigens (blue) from cells expressing Epstein-Barr virus early antigens (brown).     

Double immunoenzymatic staining for the simultaneous detection of Epstein-Barr virus induced antigens

Summary A double indirect immunoenzymatic staining was developed for the simultaneous visualization of Epstein-Barr virus-induced early antigens and virus capsid antigens in P3HR1 lymphoblastoid cell line.The double immunocytochemical staining was performed with a four-stage and a two-stage procedure employing human sera and monoclonal antibodies against Epstein-Barr virus-induced antigens, followed by the addition of specific alkaline phosphatase and peroxidase labeled antisera.The selection of substrates yielding reaction products of contrasting colours enabled the observer to distinguish cells expressing Epstein-Barr virus capsid antigens (blue) from cells expressing Epstein-Barr virus early antigens (brown).     

Transparent Golgi Impregnations: A Way to Examine Both Details of Cellular Processes and Components of the Nerve Cell Body

A method is described by which the precipitate that normally fills impregnated cells in Golgi preparations is confined to a thin and transparent scattering of fine particles that defines the somata and cellular processes. The coating is stable enough to withstand counterstaining and thus makes possible direct evaluation of structural features not only of cellular processes but also of cytoplasmic components.     

Triple staining of normal and degenerating nervous tissue.

A method of counterstaining sections impregnated according to a previously reported modification of the Glees silver impregnation is described. The basis for this counterstain is the Klüver-Barrera luxol fast blue technique. The results are illustrated and the advantages and disadvantages of the procedure are discussed.     

Gallocyanin-chrome alum counterstaining of Golgi-Kopsch impregnations

A simple technique is described for counterstaining Golgi-Kopsch impregnations. The sections are first stabilized by the method of Geisert and Updyke and then stained in 0.15% gallocyanin and 5% chromium potassium sulfate for 45 minutes at 55-60 C. The sections are then rinsed, dehydrated to 70% ethanol, cleared in terpineol, mounted and coverslipped. This procedure results in a light to medium blue stain of those cells not impregnated by the silver chromate. The major advantages of this procedure over earlier methods are: (1) the counterstain does not fade and (2) since no differentiation is required, many sections may be stained simultaneously.     

Membrane antigens of Zajdela ascitic hepatoma]

The membrane antigens of Zajdela ascitic hepatoma cells were investigated. Living cells were studied by immunofluorescence method, and solublized membrane preparations by the precipitation reacting in agar gel. Testing of the tumor cells with organospecific anti-kidney serum caused a specific fluorescence of tumor cells surface. This can be due to incorporation into the antigenic structure of the Zajdela hepatoma cell membranes of at least one organospecific antigen. Treatment of the tumor cells with organospecific anti-liver serum led to specific fluorescence of tumor cells surface. In solubilizates of the tumor cells one of the three organospecific antigens peculiar for the normal liver cells, was detected.     

Gallocyanin-Chrome Alum Counterstaining of Golgi-Kopsch Impregnations

A simple technique is described for counterstaining Golgi-Kopsch impregnations. The sections are first stabilized by the method of Geisert and Updyke and then stained in 0.15% gallocyanin and 5% chromium potassium sulfate for 45 minutes at 55-60 C. The sections are then rinsed, dehydrated to 70% ethanol, cleared in terpineol, mounted and coverslipped. This procedure results in a light to medium blue stain of those cells not impregnated by the silver chromate. The major advantages of this procedure over earlier methods are: (1) the counterstain does not fade and (2) since no differentiation is required, many sections may be stained simultaneously.     

Combining Immunodetection with Histochemical Techniques: The Effect of Heat-induced Antigen Retrieval on Picro-Sirius Red Staining

Picro-Sirius red is a routine diagnostic stain intended for the histological visualization of collagen fibers (fibrosis) in tissue. Multi-label immunohistochemistry is a powerful tool used by researchers to visualize different cell types and their location within a tissue specimen, and to observe co-localization of antigens. Combining the specificity of immunodetection with the simplicity of Sirius red staining will allow researchers to visualize multi-antigen detection in relation to fibrosis, a common histological feature of injury in many chronic diseases. Pre-treatment of formalin-fixed, paraffin-embedded tissue (FFPE) specimens with antigen retrieval is essential for the work-up of most commercially available antibodies. The most common form of antigen retrieval involves boiling tissue specimens in buffer to break the cross-linkages caused by formalin fixation. However, this method causes tissue modification and collagen fiber shrinkage leading to suboptimal results when counterstaining for Sirius red. Reduced heat and enzymatic digestion are antigen retrieval methods compatible with Sirius red counterstaining. This paper will discuss the difficulties faced when combining these two staining methods, and provide a detailed method for the simultaneous detection of antigen and Sirius red in FFPE tissues.