Counting Chromosomes in Root Tips from a Large Population of Plants

Correlative morphology of kidney tissue can be accomplished by a combination of immunofluorescent staining and light microscopy using a pre-embedding technique. Further development of this method has been elaborated by freeze-drying and chopping of the tissue, which then is incubated in biotinylated immunoglobulins. Fluorochromes and colloidal gold can be coupled to the biotinylated immunoglobulins in a second step. This technique is an alternative to postembedding immunogold staining for immunoelectron microscopy, and permits a correlative immunohistochemical and detailed morphological study of kidney tissue.     

Immuno-light and immuno-electron microscopic examination of freeze-dried kidney tissue

Correlative morphology of kidney tissue can be accomplished by a combination of immunofluorescent staining and light microscopy using a pre-embedding technique. Further development of this method has been elaborated by freeze-drying and chopping of the tissue, which then is incubated in biotinylated immunoglobulins. Fluorochromes and colloidal gold can be coupled to the biotinylated immunoglobulins in a second step. This technique is an alternative to postembedding immunogold staining for immunoelectron microscopy, and permits a correlative immunohistochemical and detailed morphological study of kidney tissue.     

A novel modification of the avidin-biotin complex method for immunohistochemical studies of transgenic mice with murine monoclonal antibodies.

When mouse tissues are probed with murine monoclonal antibodies (MAb) by indirect immunohistochemistry, the secondary antibody detects tissue-bound MAb and irrelevant, endogenous mouse immunoglobulins. The latter are a source of confounding background, especially in diseased tissues. To circumvent this problem, we generated complexes of primary MAb and biotinylated secondary antibodies in vitro for use as antigen-specific probes. After blocking free binding sites in the complexed secondary antibodies with normal mouse serum, the complexes were applied to mouse tissue sections and tissue-bound complexes were visualized with an avidin-biotin detection system. Complexes formed with 12 different rat or mouse MAb were used to probe sections of normal mice, tumor-bearing transgenic mice, and mice with tumor xenografts. The staining patterns produced by these probes reflected the specificity of the MAb in the complexes, and the labeling of irrelevant, endogenous mouse immunoglobulins was reduced substantially. This novel, indirect immunohistochemical method can be exploited to study normal and diseased mouse tissues using a variety of murine MAb.     

In situ hybridization technique using an immunogold silver staining system

Summary Anin situ hybridization technique using an immunogold silver staining detection system was used to detect biotinylated DNA probes in cultured cells and in formalin-fixed paraffin-embedded tissue. The detection method is rapid, reliable and economical, producing an insoluble signal at the site of hybridization which is visible at low-power light microscopy and which can be enhanced by epipolarization microscopy.     

Immunohistochemical localization of human immunoglobulins and lysozyme in epoxy-embedded lymph nodes: effect of different fixatives and of proteolytic digestion

The postembedding immunoperoxidase staining technique for the localization of immunoglobulins (light and heavy chains) and of lysozyme has been successfully applied to epoxy-embedded human lymph nodes, after removal of the resin. Glutaraldehyde-containing fixatives appear to be suitable for the immunohistochemical localization of human immunoglobulins and lysozyme, provided that the masked antigenicity of these proteins is recovered by proteolytic digestion of the tissue sections using 0.4% pepsin or 0.1% trypsin. Nonglutaraldehyde-containing fixatives allow the immunolocalization of human immunoglobulins without any enzymatic pretreatment. This study shows that tissues routinely fixed in glutaraldehyde and embedded for ultrastructural investigations are actually suitable for immunohistochemical studies on human immunoglobulins and lysozyme.     

Ultrastructural localization of intracellular immunoglobulins in Epon-embedded human lymph nodes. An immunoelectron microscopic investigation using the immunogold staining (IGS) and the avidin-biotin-peroxidase complex (ABC) methods

The ultrastructural localization of intracellular immunoglobulins on ultrathin sections of glutaraldehyde-fixed, postosmicated, and Epon-embedded human lymph nodes has been achieved using such highly sensitive immunocytochemical techniques as immunogold staining and avidin-biotin-peroxidase complex. These immunoelectron microscopic techniques allow the identification of intracellular immunoglobulins without affecting the ultrastructural morphology of the tissue, since they do not require any pretreatment of the sections with proteolytic enzymes or deresinating agents. Therefore, immunoglobulins can be precisely localized in the cell organelles; structures whose morphology is well preserved. The availability of a reliable postembedding staining procedure for the ultrastructural localization of immunoglobulins is of definite value for investigations on human lymphoid tissue, both normal and pathological.     

Immunohistology on formol-sublimate fixed and paraplast embedded kidney tissue with comparison to formalin fixation and to pre-embedding immunofluorescent staining

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 micron Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.     

Demonstration of immunoreactive sites on cartilage after in vivo administration of biotinylated anti-type II collagen antibodies

Administration of biotinylated monoclonal antibodies provides the basis of a simple technique for identifying immunoreactive sites in vivo. Biotinylated anti-type II collagen antibodies were injected intraperitoneally into normal DBA/1 mice. The mice were sacrificed after 96 hr and the front paws removed and decalcified to allow tissue sectioning before snap-freezing. Binding of antibodies in vivo was visualized with affinity cytochemical staining using avidin-biotin-peroxidase complexes. Specific binding of antibodies to cartilaginous structures was seen after injection of 20-500 micrograms biotinylated monoclonal or polyclonal anti-type II collagen antibodies, but not after injection of a biotinylated control antibody. This technique should further the detection and localization studies of tissue components involved in the dynamics of physiological and pathological processes.     

Detection of plasma cell immunoglobulins in tissue sections optimally fixed for ultrastructural immunocytochemistry

We describe the ultrastructural localization of plasma cell immunoglobulins in vibratome sections of popliteal lymph nodes. Fixation with glutaraldehyde-paraformaldehyde gave better tissue and antigen preservation than paraformaldehyde or periodic acid lysine-paraformaldehyde; biotinylated Fab fragments of sheep anti-mouse IgG-streptavidin-biotinylated horseradish peroxidase (HRP) or Fab-HRP conjugates gave similar results. With both immunoreagents, excellent tissue preservation and antigen detection was observed in the first layer of cells sectioned with the vibratome. Conjugates of anti-mouse IgG with HRP did not show any staining. Peroxidase stain was observed in the nuclear envelope, cisternae of the rough endoplasmic reticulum, and the Golgi apparatus complex. In the Golgi apparatus, staining was seen consistently in cisternae of the cis face and in adjacent vesicles; the trans cisternae showed weak or no stain, and adjacent vesicles, "coated" vesicles, and granules were not stained. This study shows that high quality of tissue preservation and antigen detection, by both light and ultrastructural immunocytochemistry, is feasible in tissue fixed with glutaraldehyde-paraformaldehyde followed by vibratome sectioning and immunostaining with Fab-biotin-streptavidin-biotin-HRP, or Fab-HRP.     

Immunohistology on Formol-Sublimate Fixed and Paraplast Embedded Kidney Tissue with Comparison to Formalin Fixation and to Pre-Embedding Immunofluorescent Staining

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 μm Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.     

The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens.

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimide-biotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs.     

Mixed glycosidase pretreatment reduces nonspecific binding of antibodies to frozen tissue sections

Indirect immunohistochemical studies of frozen mouse tissues with mouse monoclonal antibodies yield, in general, suboptimal results primarily because of indiscriminate binding of secondary antibody to all mouse immunoglobulins, i.e., to the monoclonal reagent and to endogenous immunoglobulin nonspecifically trapped in the tissue. To reduce this nonspecific staining, frozen sections of mouse kidney were treated enzymatically. Optimal results were obtained following a 2 hr treatment with 20 mg/ml of mixed glycosidases (MG). This treatment reduced the nonspecific background staining of the interstitial spaces and blood vessels, but did not affect the reactivity of structurally bound immunoglobulin G (IgG) in the glomeruli or alter the reactivity of mouse renal tissue to the monoclonal antibody that recognizes an oligosaccharide antigenic determinant (SSEA-1). Eluates from enzyme-treated frozen tissue sections contained normally immunoreactive IgG in the form of dimers. These data indicate that MG treatment of frozen sections could be safely used to reduce the content of nonstructurally bound immunoglobulins in frozen tissues and thus improve the visualization of specific monoclonal antibody binding.     

Immunohistochemical identification of tubular segments in percutaneous renal biopsies

Summary To identify the renal cortical tubular segments involved in tubulo-interstitial disease in formalin-fixed, paraffin-embedded percutaneous kidney biopsies, we developed multiple immunolabeling protocols using segment-specific tubular markers. The present study of biopsies from patients with minimal change or thin basement membrane nephropathy provides a baseline for interpretation of histopathology. Proximal tubules were stained either by the PAS reaction or by the biotinylated Phaseolus vulgaris erythroagglutinin (PHA-E)-streptavidin-gold-silver system (brush borders black). The anti-Tamm-Horsfall (THP) antibody-immunoperoxidase (aminoethylcarbazole, AEC-IPO), and anti-epidermal cytokeratins (ECK) antibodies-immunoalkaline-Fast Blue BB methods marked the distal straight tubules and the cortical collecting system red-brown and blue, respectively. When these immunolabelings were combined, the coapplication of AEC-PO-labeled peanut agglutinin (PNA) or anti-epithelial membrane antigen antibody-AEC-IPO technique (both are markers for distal nephron) visualized the apical membranes of distal convoluted tubules. In the protocol PHA-E + PNA + THP + ECK, the tubular basement membranes were outlined by the anti-laminin antibody-AEC-IPO staining, carried out simultaneously. The protocol PNA + THP + ECK + PAS was found to be a quite appropriate multiple immunolabeling method for the tubules, and is recommended for use as a tool in the study of tubulo-interstitial diseases.Abbreviations PAS periodic acid-Schiff reaction - PHAE Phaseolus vulgaris erythroagglutinin - PNA Peanut agglutinin - EMA epithelial membrane antigen - THP Tamm-Horsfall glycoprotein - ECK epidermal cytokeratins - PO peroxidase - Biot-PHA-E biotinylated PHA-E - APAAP complexes of alkaline phosphatase and mouse monoclonal anti-alkaline phosphatase - SWARI swine anti-rabbit immunoglobulins - FCS fetal calf serum - TBS Tris-buffered saline - AEC aminoethylcarbazole - DAB diaminobenzidine - FBBB Fast Blue BB - IA immunoalkaline - GL glomerulus - PT proximal tubule - DST distal straight tubule - DCT distal convoluted tubule - CCS cortical collecting system - CT connecting tubule - CD collecting duct     

The use of avidin-gold complex for light microscopic localization of lectin receptors

Summary In the present study, we have investigated the use of avidin-gold complex (AG) as a possible cytochemical marker for visualizing and identifying lectin receptors in deparaffinized tissue sections. Monodispersed gold sols of 15 nm average diameter were prepared by sodium citrate reduction. The AG complex was prepared with highly purified egg-white avidin (avidin-D).Deparaffinized sections of cat duodenum were labeled with five different biotinylated lectins, then were washed and stained for 1–2 h with AG. Intensification of the gold staining was achieved by a modification of the silver-enhancement method. For each lectin, the labeling properties of the avidin-gold-silver (AGS) were compared with those of the avidin-biotin-peroxidase (ABC) and the lectin-gold (LG) methods. We found the lectin binding pattern demonstrated by the AG method to be similar to that of the ABC. The AG localization of the carbohydrate residues is more precise, compared to the peroxidase reaction due to lack of diffusion of the gold marker. Labeling with AGS resulted in improved staining over the AG method, similar to the staining intensity of the ABC. In addition, the two-step AG method provided more intense staining than the direct one-step procedure of the lectin-gold labeling.In conclusion, the use of the AGS method for histochemical visualization of lectin receptors requires a simple two-step procedure which allows highly accurate localization of tissue glycoconjugates. It entails using only a single gold-ligand complex applicable to any biotinylated lectin regardless of its biochemical nature. It can also be easily adapted for use with other biotinylated ligands such as antibodies, hormones, toxins, etc.     

Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates

The use of fluorescein-avidin or rhodamine-avidin conjugates in conjunction with biotinylated secondary antibodies for indirect immunohistology frequently results in pronounced nonspecific nuclear staining in kidney sections. This nonspecific nuclear staining can be effectively blocked by using 5% (w/v) nonfat dry milk in buffered saline as the diluent for the avidin conjugates. In contrast, 5% (w/v) bovine serum albumin, a commonly used blocking agent, has only a modest effect. Nonfat dry milk is also effective as a blocking agent and carrier when used in fluorescence-activated cell sorter (FACS) analysis. These results emphasize the broad usefulness of milk-based blocking reagents.     

Use of avidin for speedy clearance of biotinylated immunoglobulins from the blood flow

The phenomenon of a hundredfold more rapid blood clearance of biotinylated immunoglobulins after post-injection of an equiponderate dose of avidin is described. The concentration of 125I-labeled biotinylated IgG in rat circulation slowly decreased to 20% of the initial level in 24 hours. Avidin injection at any moment of this period induced a 90-95% reduction of blood radioactivity in 15 minutes. Up to 70% of the radioactivity was recovered in the liver. The technique of enhanced blood clearance developed in rats was checked in dogs using biotinylated monoclonal anti-human fibrinogen antibodies, capable of concentrating in dog thrombus. The results obtained offer the possibility of thrombus/blood contrast increase in radioimmunoimaging.     

A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling.

We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.     

Biotinylated epidermal growth factor: a useful tool for the histochemical analysis of specific binding sites

Summary Epidermal growth factor (EGF) was labelled with biotin via modification of either the amino or carboxyl groups, using suitable reagents, namely biotinyl-N-hydroxysuccinimide ester or biotinamidocaproyl hydrazide. To assure that the specific binding capacity of EGF is retained despite its chemical modification, displacement of the EGF by biotinylated derivatives in a routine binding assay was performed. The inhibitory potency compared to unmodified EGF was only slightly reduced. This result is the prerequisite for testing the usefulness of biotinylated EGF in histochemistry. The biotinylated probes were applied to sections of human tumour tissue and of monkey organs (liver, kidney, uterus of Cynomolgus and Rhesus monkey) to localize the specific binding sites for EGF. Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and incubated with the probes at a concentration of 10 g ml^–1 at room temperature for 60 min. Specific binding of the EGF was visualized by the avidin-biotin techniques (ABC). A positive reaction in conjunction with appropriate controls by competitive inhibition was seen for all monkey tissue sections and for the following number of cancer cases: breast carcinoma: 7/10; mesothelioma: 2/4; lung carcinoma: 1/3; colon carcinoma: 1/3.The staining properties were similar for both types of probes that differed in the functional group that is involved in modification by biotion attachment. However, the batches with modification of the amino groups stained more intensely and more distinctly than the carboxyl modified EGF. Overall, the data indicate that the ligand properties of the EGF are not impaired by biotinylation of the two types of functional groups. Thus, biotinylated EGF is a useful tool for histochemical detection and identification of EGF specific binding sites in mammalian tissue.     

A novel immunohistochemical technique for demonstration of specific binding of human monoclonal antibodies to human cryostat tissue sections

We describe a novel immunohistochemical technique which permits the detection of specific binding of human monoclonal antibodies (MAb) to cryostat sections of human tissues. The technique overcomes the problem of background staining caused by the presence of endogenous immunoglobulins in tissue sections. This is achieved by the formation of a molecular complex of the primary antibody (a human MAb), horseradish peroxidase-conjugated goat anti-human immunoglobulin, and normal human serum. This complex is then incubated with cryostat sections of human tissue, and binding of the complex is demonstrated using diaminobenzidine/hydrogen peroxide. The method is suitable for immunohistochemical screening of small samples of tissue culture supernatant for the presence of human MAb of potential interest, and for determining the pattern of binding of such MAb to a wide range of normal and pathological human tissues.     

Binding properties of biotinylated epidermal growth factor to its receptor on cultured cells and tissue sections

A biotinylated derivative of murine epidermal growth factor (EGF) was prepared by covalent attachment of the terminal amino group of EGF to N-biotinyl-epsilon-aminocaproyl-N-hydroxysuccinimide. The stoichiometry of biotin incorporation was in the range of one biotin moiety per EGF molecule. The biotinylated EGF (biotinyl-epsilon-caproyl-EGF, BioEGF) binds to EGF receptors on intact Ehrlich ascites carcinoma (EAC) cells with an affinity similar to that of native EGF and displays the same mitogenic activity as EGF in a soft agar test system with normal rat kidney (NRK) cells. BioEGF was visualized on cultured cells and tissue sections of a head and neck tumour by commercial streptavidin/avidin detection systems. Cytochemical analyses of certain tumour forms can be easily performed using the BioEGF probe.