Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.

A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37 degrees C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.     

Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues

Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidin-biotin-peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin-Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol-fixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanol-fixed tissues. The distribution of the positive nuclei in the two latter cases coincided with the proliferative compartment. On the other hand, double labelling with [3H]-thymidine and with the cyclin/PCNA antibody revealed that in methanol-fixed tissues the cyclin/PCNA labelling index did not differ by more than 6% from the [3H]-thymidine index. Besides the two labels overlapped in a proportion of labelled cells that was in reasonable agreement with expectation considering cells flow in and out of S phase since the time of [3H]-thymidine injection. This indicates that both labels recognize the same cells in this material. In contrast, in formaldehyde-fixed tissues, the cyclin/PCNA labelling index markedly exceeded the [3H]-thymidine labelling index. From this it is concluded that cyclin/PCNA immunostaining can be used: (1) In formaldehyde-fixed tissues (including existing material stored as paraffin blocks): for defining and mapping the proliferative (or germinative) compartment. (2) In methanol-fixed tissues as a substitute to the [3H]-thymidine autoradiographic labelling index. From this, a method is proposed (derived from classical 'double-labelling' technique) for measuring S phase duration in tissues fixed at a known interval time after a single labelling with [3H]-thymidine (or BrdU) and submitted to cyclin/PCNA immunocytochemical detection and to autoradiography (or to BrdU immunostaining).     

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion

Summary A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37° C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.     

Immunohistological optimization of detection of bromodeoxyuridine-labeled cells in decalcified tissue

Mice were injected with a range of bromodeoxyuridine (BrdU) concentrations from 0.01 mg to 10 mg, and their jaws were fixed in buffered formalin or modified Carnoy. After EDTA or formic acid decalcification, a range of DNA denaturation schedules was assessed and immunohistological detection of BrdU-containing nuclei was performed using the Sera Lab anti-BrdU antibody MAS 250b. For Carnoy-fixed tissue, denaturation in 1 N HCl for 8 min at 60 degrees C was capable of adequately detecting an injected dose of 0.05 mg but not a dose of 0.01 mg BrdU, whereas pepsin/HCl treatment gave only weak staining after injection of 1 mg BrdU. In comparison, formalin fixation required pre-treatment with 0.2-0.4% pepsin/HCl at 37 degrees C for comparable staining intensity, but could still not adequately detect a dose of 0.1 mg BrdU. There was little detectable difference in staining between EDTA- and formic acid-decalcified tissues after injection of 10 mg BrdU.     

Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues

Abstract Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidin-biotin-peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin-Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol-fixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanol-fixed tissues. The distribution of the positive nuclei in the two latter cases coincided with the proliferative compartment. On the other hand, double labelling with [³H]-thymidine and with the cyclin/PCNA antibody revealed that in methanol-fixed tissues the cyclin/PCNA labelling index did not differ by more than 6% from the [³H]-thymidine index. Besides the two labels overlapped in a proportion of labelled cells that was in reasonable agreement with expectation considering cells flow in and out of S phase since the time of [³H]-thymidine injection. This indicates that both labels recognize the same cells in this material. In contrast, in formaldehyde-fixed tissues, the cyclin/PCNA labelling index markedly exceeded the [³H]-thymidine labelling index. From this it is concluded that cyclin/PCNA immunostaining can be used:

• 1 In formaldehyde-fixed tissues (including existing material stored as paraffin blocks): for defining and mapping the proliferative (or germinative) compartment.
• 2 In methanol-fixed tissues as a substitute to the [³H]-thymidine autoradiographic labelling index.

From this, a method is proposed (derived from classical ‘double-labelling’technique) for measuring S phase duration in tissues fixed at a known interval time after a single labelling with [³H]-thymidine (or BrdU) and submitted to cyclin/PCNA immunocytochemical detection and to autoradiography (or to BrdU immunostaining).     

Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.     

Ethidium bromide: a nucleic acid stain for tissue section

The phenanthridinium dye, ethidium bromide (EB), selectively intercalates into double-stranded regions of nucleic acids with a large and specific increase in fluorescence. When used for the staining of fixed tissue sections, the dye stains cellular nuclei with excellent resolution of microscopic detail. In some fixed tissues, particularly pancreatic acini, cytoplasm stains intensely and this staining can be abolished by digestion with trypsin and ribonuclease. The orange fluorescence of EB can be easily distinguished from the green fluorescence of fluorescein and EB is thus an excellent counterstain for immunofluorescence. Ethidium bromide is a useful and practical stain for the fluorescence microscopy of tissue sections and, in combination with enzymatic digestion of RNA, provides a simple way to differentially localize DNA and RNA.     

Specific demonstration of ribonucleic acid by chemical bromination and immunohistochemistry

In this report we describe a specific staining procedure for detection of ribonucleic acid (RNA), based on bromination of uracil and subsequent immunohistochemical visualization of 5-bromouracil in RNA. This method is applicable for both cryostat and glycol methacrylate (GMA)-embedded sections. Cryostat sections must be fixed in formaldehyde, whereas tissue pieces to be embedded in GMA are fixed in cold acetone. Before bromination, sections must be treated with trypsin. Bromination was performed in a solution of bromine in potassium bromide. After bromination, excess bromine was removed with sodium bisulfite. The monoclonal antibody MoBu-1 specifically bound to brominated RNA. Ribonuclease digestion, in contrast to deoxyribonuclease digestion, abolished staining. This method makes possible precise localization of RNA, especially well demonstrated in plastic-embedded sections.     

A rapid immunogold-silver staining for detection of bromodeoxyuridine in large numbers of plastic sections, using microwave irradiation

Summary A rapid and convenient method for the large scale, immunogold-silver staining (IGSS) of bromodeoxyuridine (BrdU) incorporated by S phase cells, by means of a monoclonal antibody (anti-BrdU) is described. Nineteen slides at a time can be incubated with the antibodies and the protein A-gold (PAG) in staining jars. The antibody and protein A-gold solutions could be used at least five times to incubate new batches of slides. The incubation times with these solutions were shortened by means of microwave irradiation. In this way 200 slides carrying at least 800 sections could be easily processed under the same conditions in one day, using 1.25ml neat antibody solutions of anti-BrdU and rabbit anti-mouse.For light microscopy bothpplastic embedding systems: methylmethacrylate (MMA) and glycolmethacrylate (GMA) can be stained with this technique. The MMA sections, of which the plastic has to be removed before the IGSS, has the advantage of a stronger labelling intensity. The GMA plastic, which contains a cross-linking, agent cannot be removed and consequently for GMA sections it is necessary to incubate the sections with a proteolytic enzyme (trypsin) before the IGSS, to reexpose the antigenic binding sides. However, the GMA sections can be allowed to air dry during the IGSS without negative effects on the morphology. This makes it possible to perform the antibody and the PAG-incubating steps on one day and to finish the IGSS the next day. In this way twice as many GMA slides can be incubated with the same antibody and PAG solutions than with MMA slides.In both plastic embedding systems the intensity of the BrdU labelling was found to be stronger in Carnoy's than in Bouin's fixed sections.     

Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathologic study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tissue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.     

Collagenase in the immunohistochemical demonstration of laminin, fibronectin and factor VIII/RAg in nervous tissue after fixation

The effects of collagenase on the immunohistochemical demonstrability of laminin, fibronectin and Factor VIII/RAg in human nervous tissue have been studied. The influence of this, and other proteolytic enzymes such as pepsin and trypsin, has been investigated in relation to different fixatives. Collagenase gave better results with Carnoy fixed material than after formalin fixation; unlike trypsin and pepsin, it did not produce tissue digestion.     

Influence of fixatives on the immunoreactivity of paraffin sections

Summary The unlabelled antibody peroxidase-antiperoxidase (PAP) technique was used to demonstrate IgA in paraffin sections of human tonsil fixed in eight commonly known fixatives. In all but one of the fixatives, treatment with trypsin prior to immunostaining resulted either in digestion of sections, or no change in positive staining. In tissues fixed in neutral buffered formalin-saline, positive staining was absent unless the sections had been treated with trypsin, and it has been shown that digestion requirements of different areas throughout a section may vary according to the effectiveness of fixation.     

Microwave-aided technique to detect bromodeoxyuridine in S-phase cells using immunogold-silver staining and plastic-embedded sections

Summary A technique is described to detect bromodeoxyuridine (BrdU) incorporate by cells in S-phase, with a monoclonal antibody, using removable plastic embedding and immunogold-silver staining (IGSS). The incubation times were reduced and the immunological reactions enhanced by microwave irradiation.The embedding in methyl methacrylate enabled us to make thinner sections and it improved the quality of the preparations. The methyl methacrylate did not hinder the reaction of BrdU with the antibody because it could be removed prior to the IGSS procedure. The IGSS procedure appeared to be very sensitive, requiring lower concentrations of the antibodies than other methods. The use of microwave irradiation shortened the time needed to stain a section from 7 to less than 4 h. Furthermore, using microwave irradiation, the concentration of the antibodies needed could be reduced even further compared with the normal IGSS procedure.In sections of the mouse testis and small intestine only nuclei of cells known to be able to proliferate appeared BrdU positive. The non-specific background staining was found to be negligible. In testes of mice that received both³H-thymidine and BrdU more than 95% of the radioactively labelled cells also showed BrdU label and vice versa. This indicates that both methods are equally sensitive for detecting cells in S-phase.     

New technique for processing paraffin-embedded tissue for Y chromosome fluorescence identification of trophoblastic disease

A new reprocessing technique for Y chromosome fluorescent body (q 12 region) detection of trophoblastic disease in previously paraffin-embedded tissues is described. Deparaffinized sections were treated with pronase and trypsin for digestion, followed by hydrolysis with HCl and acetic acid, staining with quinacrine hydrochloride fluorochrome and mounting in S?rensen's phosphate buffer (pH 5.5). Use of the technique resulted in sufficient fluorescence quality and better accuracy for Y and X heterochromatin scoring. The technique yielded the same results in retrospective formalin-fixed, paraffin-embedded trophoblastic specimens as in fresh tissues. The combinations of enzymes and acids and the dosages necessary for optimal results are discussed.     

De-staining and re-staining mucins in formalin fixed paraffin sections

Abstract

Re-staining of formalin fixed paraffin sections sometimes is required and this requires prior de-staining. Some simple and effective protocols for de-staining are described. Mucihematoxylin and mucicarmine can be removed with acid alcohol. Zirconyl hematoxylin can be removed with periodic acid or Sinha's fixative. Alcian blue can be removed with 5% trifluoroacetic acid in dichloromethane. Colloidal iron can be bleached in 1% household bleach in alcohol. PAS can be removed with hydrogen peroxide or ammonium hydroxide. With few exceptions, de-stained sections can be re-stained with mucihematoxylin, PAS or Gabe's trichrome.     

Detection of S-Phase Cells in Smear Cytology Using in Vitro Bromodeoxyuridine Labeling

A technique Is described for rapid detection of S-pha?e cells of tumor tissues in smear specimens using bromodeoxyuridine (BrdU) immunostaining. Mouse NR-S1 tumors and human tumor specimens were prepared for smear cytology after incubation in RPMI 1640 culture medium containing 200 μM BrdU at 37 °C under 3 atm for 1 hr. Samples were fixed in 70% ethanol for 30 min and used immediately or air dried for 30 min. Samples were then denatured in either 4 N HC1 or 0.07 N NaOH to prepare partially single-stranded DMA. Fixation with air drying for 30 min followed by 30 min in 70% ethanol and 1 min denaturation with 0.07 N NaOH resulted in satisfactory staining quality. Cultured tumor specimens were processed for routine paraffin sections after smears were made for cytology. The labeling indices of the smear specimens and of the paraffin sections gave similar results. This technique should be useful in evaluating the cell proliferative potential of tumor tissue in smear cytology without processing paraffin sections.     

The histochemistry of asparagine-linked oligosaccharides of glycoproteins in mammalian and avian tissues as studied by digestion with almond glycopeptidase

Summary In a series of mammalian and avian tissues, asparagine-linked oligosaccharides of glycoproteins have been localized histochemically by mearis of combined periodic acid-Schiff (PAS) and almond glycopeptidase digestion procedures. According to the present results, the particular saccharide residues were found to be localized primarily in glycoproteins of connective and muscular tissue elements but were hardly visualizable or absent in glycoproteins of epithelial tissue elements. In view of the substrate specificity of almond glycopeptidase and the known staining selectivity of the PAS reaction, the majority of connective and muscular tissue elements are thought to be the main sites where plasma types of glycoproteins are involved.     

The Action of Ribonuclease on Fixed Tissues

To study the optimal conditions for histochemical use of ribonuclease on fixed tissues, the factors of (1) type of fixation, (2) temperature, pH, type of buffer and length of incubation, (3) concentration of enzyme, and (4) staining and dehydration of sections were observed on rabbit pancreas.

The fixing fluids studied were sublimate-alcohol, Bouin's, Zenker-acetic, Zenker-formol, Petrunkevich's cupric-paranitrophenol, 10% neutral formalin, SUSA, Carnoy, Bensley's chrom-sublimate, absolute ethyl alcohol and acetone. Formaldehyde was a satisfactory fixative, although others might be preferred for special purposes. Of the five buffers tested, McIlvaine's citric-acid-disodium-phosphate mixture was the most satisfactory, whereas veronal-acetate extracted considerable stainable cytoplasmic material. The optimum concentration of ribonuclease and length of incubation varied greatly after the 11 different types of fixation. For example, with ribonuclease buffered by Mcllvaine's fluid, the intense cytoplasmic staining of formaldehyde-fixed tissues was removed by concentrations as low as 0.001 mg./ml., whereas, with sections fixed in Zenkers fluid some cytoplasmic staining persisted even after 3 hours in 0.2 mg./ml. Under the conditions employed the temperature and hydrogen-ion concentration during incubation were less important. Examples of nonspecific action of ribonuclease were noted. Until the degree and optimum conditions of specific action have been more precisely established by further experiments, it is suggested that this histo-chemical reaction only be interpreted as a confirmatory test which is, under the best conditions, only relatively specific for ribonucleic acid and not highly quantitative.     

Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.     

Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.