Heterogeneous staining of rat hepatocyte nuclei by HBFP technique

Synopsis The non-enzymatic histochemical technique Haematoxylin-Basic Fuchsinpicric acid (HBFP) was studied in fresh-frozen and Carnoy-fixed, paraffin-embedded rat liver sections. The hepatocyte nuclei fell into two populations and showed either a crimson red or purple staining in frozen as well as paraffin sections. The heterogeneous staining of the rat heptocyte nuclei was also present when the tissue sections were stained by Methyl Green-Pyronin stain. The differing nuclear staining was present in the isolated nuclei also. The HBFP technique, therefore, appears potentially useful when applied to liver and other tissues as well.     

Comparison of monoclonal antibodies PC 10 and MIB 1 on microwave-processed paraffin sections

Abstract. Antibodies against the proliferation-associated nuclear antigen (PCNA) and against the Ki-67 protein are widely used as operational proliferation markers in human tumour diagnostics. The original Ki-67 antibody had the inherent drawback in that it could only be used when fresh-frozen material was available. The antibody PClO was supposed to offer the advantage that it could be applied on formalin-fixed and paraffin-embedded tissues. However, in cases in which the formalin fixation exceeded 4 h, PC10 staining proved to be inconsistent and often failed.
The aim of this study was to compare a recently prepared Ki-67 equivalent monoclonal antibody (MIB 1) and PC10 in routinely fixed histopathological material using antigen retrieval by microwave processing.
Antibody MIB 1 stained the nuclei of cells known to belong to the proliferative compartments in microwave-processing paraffin sections of formalin-fixed tissues. Quiescent cells were consistently negative for MIB 1 staining. In contrast, PC10 was positive in almost all nuclei of different tissues in microwave-treated paraffin sections. Thus, antigen retrieval by microwave processing is beneficial for the detection of the Ki-67 protein in paraffin sections, whereas it is not needed for the detection of the PCNA.     

Compared intracellular localization of the glucocorticosteroid and progesterone receptors: an immunocytochemical study

The intracellular distribution of the glucocorticosteroid and progesterone receptors (GR and PR, respectively) was studied immunohistochemically. In control adrenalectomized (Adx) rat liver, immunostaining of paraffin sections revealed GR in cell nuclei, with a wide range of intensity between individuals. Following dexamethasone (Dex) treatment, the nuclear staining was uniformly high in all animals; the cytoplasmic staining was always weak and remained unchanged after Dex treatment. In frozen sections, the GR immunoreactivity in cell nuclei was weak in the absence and very strong in the presence of Dex, while no GR-specific cytoplasmic staining was observed. In frozen sections fixed in vapor of formaldehyde to avoid any artifactual redistribution of the receptor, some GR immunostaining was observed in the cytoplasm and the nucleus. In contrast, in paraffin as well as in frozen sections of chick oviduct, fixed by immersion or in vapor, PR was exclusively nuclear, including in the absence of progesterone, and the intensity of immunostaining was not modified by progesterone treatment. In order to verify if loss of nuclear receptors during tissue preparation could explain the differences in nuclear immunostaining observed between hormone-free and hormone-occupied GR, and between GR and PR, frozen sections of Adx rat liver and chick oviduct were preincubated at 4 degrees C in buffer solutions before the fixation procedure. It was found that hormone-free GR diffused out of the nucleus faster than hormone-occupied GR nuclei, and that nuclear GR diffused faster than nuclear PR. Based on these results, we propose that, during the fixation procedure, the fraction of nuclear GR which diffuses out of the nucleus is much smaller in the presence than in the absence of Dex. This lesser loss of nuclear GR after Dex treatment results in an increase of immunostaining after hormonal administration, which might have been erroneously interpreted as a sign of translocation from cytoplasm to nucleus. That the nuclear PR detection is not modified by progesterone treatment may be explained by its reduced diffusibility as compared to nuclear GR. This hypothesis does not rule out the existence of some cytoplasmic GR, whose significance remains unclear, but it offers a unified mechanism of action for all steroid hormone receptors. In the case of glucocorticosteroids, as already proposed for estradiol and progesterone, no step of cytoplasm to nucleus translocation would be required for hormone action, and transformation-activation would occur in the nucleus, resulting in tighter binding of the hormone receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric analysis of estrogen receptor expression in isolated nuclei and cells from mammary cancer tissues.

BACKGROUND: Cellular expression of receptors for the hormones estrogen and progesterone in human mammary tumors is of diagnostic and prognostic value. Ligand binding assays have been replaced by immunohistochemical analysis of receptor expression. However, both of these techniques are slow, and in the ligand-binding assay it is difficult to measure heterogeneity of receptor expression in individual cells. Flow cytometry has been used extensively for monitoring the expression of cellular receptors in hematopoietic tumors but has been of limited value in the analysis of mammary tumors, which are difficult to disaggregate into single cells for flow analysis. Hormone receptors have a predominant nuclear localization, and it is relatively easy to isolate nuclei from paraffin-embedded archival tissues for flow cytometric analysis of receptor expression. METHODS: Thick sections from formalin-fixed paraffin-embedded archival mammary tumors were digested by different enzyme solutions for the isolation of single nuclei. Different fixatives were used to compare the results on subsequent staining of the nuclei for estrogen receptor (ER) expression. Double staining with propidium iodide and fluorescein isothiocyanate labeled secondary antibodies for ER expression was used for multiparametric analysis of ER and DNA content. RESULTS: Digestion of paraffin sections with low concentration of pepsin and detergents was ideal for isolation of single nuclei. Fixation with paraformaldehyde in the presence of Triton X-100 improved staining of the cells. Isolated nuclei had enhanced immunoreactivity compared with the whole cells, and subpopulations differing in reactivity could be identified in the nuclear fractions. Double staining of nuclei for ER expression and DNA content could allow for multiparametric analysis of these two important parameters. CONCLUSIONS: The procedures described can be used for processing of archival paraffin-embedded mammary tumors for monitoring of ER expression and aneuploidy. These two parameters have important diagnostic and prognostic significance in mammary tumors. Laser flow cytometry by providing multiparametric analysis can allow for correlation of these cellular markers with other important cellular and clinical parameters.     

Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut

We tested four synthetic substances for their histochemical value to demonstrate the catalytic activities of chymase or tryptase in mast cells in sections of human gut. Both Suc-Ala-Ala-Phe-4 methoxy-2-naphthylamide (MNA) and N-acetyl-L-methionine-alpha-naphthyl ester (alpha-N-O-Met) reacted with chymase but not tryptase in mast cells. Conversely, D-Val-Leu-Arg-MNA and Z-Ala-Ala-Lys-MNA were hydrolyzed by mast cell tryptase but not chymase. These results were confirmed by use of two inhibitors of chymotrypsin-like activity, chymostatin and Z-Gly-Leu-Phe-chloromethyl ketone (CK) and two inhibitors of trypsin-like activity, Tos-Lys-CK and D-Val-Leu-Arg-CK. Excellent staining reactions were obtained on cryostat sections of unfixed or aldehyde-fixed tissues and on paraffin sections of Carnoy-fixed tissues. For chymase, however, Suc-Ala-Ala-Phe-MNA is preferred on cryostat sections because it is more specific. On paraffin sections alpha-N-O-Met is preferred because other cells are not then stained. For tryptase, Z-Ala-Ala-Lys-MNA was more selective and more specific and is the preferred general purpose substrate on cryostat sections of aldehyde-fixed tissues and for paraffin sections. D-Val-Leu-Arg-MNA is the preferred substrate for cryostat sections of unfixed tissue. Only a limited number of mast cells showed a reaction for chymase, and these occurred mainly in the submucosa. All mast cells, however, gave a reaction for tryptase, and we recommend the use of either substrate for this enzyme for routine detection of mast cells in human tissues. Double staining for the two main mast cell proteases is most conveniently undertaken on paraffin sections of Carnoy-fixed tissues using MNA substrates for tryptase and alpha-N-O-Met for chymase.     

Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the Methyl Green-Pyronin technique with the Gallocyanin chromalum and Feulgen procedures using image cytometry

Summary For simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA.The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen analtion. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm.After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiff's reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum.We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine.     

Improved Histology for the Chick-Quail Chimera

A simple modification of nuclear staining after acid hydrolysis has been made which provides easy identification of quail nuclear markings in a chick-quail chimera. This method also improves the histologic detail normally seen with hematoxylin and eosin when compared to the more commonly used Feulgen reaction. Embryonic tissues can be fixed in Zenker's or Helly's solution and the sections obtained are hydrolyzed in acid (3.5 N HCl at 37 C for 40-50 min). After acid hydrolysis the sections are stained with hematoxylin and eosin rather than Schiff reagent and fast green. The interphase nuclei of chick cells show homogeneous or mottled purplish blue staining, while quail nuclei contain a dark blue spot. This staining corresponds to the reddish purple staining of the quail's heterochromatin seen adjacent to the nucleolus in the standard Feulgen stain. This new technique facilitates identification of quail cell types in the chick host and provides superior histology of the chick tissues by demonstrating cytoplasmic detail.     

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

The Histochemical Detection of iron in Tissues

The fixation and staining of iron in tissues is discussed. Procedures for demonstrating iron in hemoglobin and nuclei are also briefly considered.

Lillie's formalin buffered at neutrality gave the optimal fixation for iron. The Prussian blue method was preferred to the Turnbull blue. Lison's procedure of the former, slightly modified, gave the most satisfactory results. When a procedure is required that employs non-iron-containing reagents, Macallum's or Mallory's hema-toxylin and Quincke's ammonium sulfide method are useful. The former, though not entirely specific, is preferable under controlled conditions when the quantities of iron are small. Hemoglobin iron in paraffin sections can be demonstrated by the usual procedures for iron after previous exposure of the section to peroxide, as recommended by Brown. The property of nuclei to adsorb iron from inorganic sources and from hemoglobin can readily be shown; caution is required in interpreting the iron detected in nuclei after Macallum's sulfuric acid hydrolysis.     

Romanowsky-type staining: Enzymatic analysis of nuclear metachromasia in formalin-fixed paraffin sections

The red color of nuclei produced in formol-fixed paraffin sections stained with toluidine blue has been investigated by using deoxyribonuclease (DNase), ribonuclease (RNase) and 0.1 M Tris buffer. The action of DNase on formol-fixed material is not fully reliable, but clear-cut when positive. Nuclear basophilia and metachromasia is removed, nucleolar and cytoplasmic RNA is preserved. The picture produced by RNase depends to some extent on the concentration and acidity of the toluidine blue used for subsequent staining. Cytoplasmic RNA is always removed, while the red stain in nuclei usually remains intact. With 0.1% toluidine blue in 1% acetic acid, a nuclear color change from red to pale green is observed. Using this same staining solution, it can be shown that 0.1 M Tris buffer (overnight extraction at 37° C) will remove cytoplasmic RNA but leave intact the nuclear material that stains red. A red to green shift can subsequently be produced by RNase. From this it is deduced that there is a chromatin-associated nuclear RNA fraction which can be removed by the enzyme, but is stable to the buffer solution.     

A restaining method to restore faded fluorescence in tissue specimens for quantitative confocal microscopy.

The aim of this study was to develop a procedure to remove the TO-PRO-3 fluorescent dye from tissue sections and restain with TO-PRO-3, still allowing calculation of DNA content and distribution by confocal laser scanning microscopy (CLSM). This would allow repeated measurements on the same tissue sections and prevents loss of tissue material from valuable clinical samples. Thick sections (14 microm) were cut from a paraffin block of adrenal tissue and stained using TO-PRO-3. Image stacks were acquired by CLSM. Thereafter, three destaining approaches were tested based on incubation, at different temperatures and durations, in the medium that is normally used to dissolve TO-PRO-3. The same areas were imaged again to measure residual fluorescence and were subsequently restained and imaged again. The intensity of the images acquired after initial staining and restaining were compared. A number of 3-D (texture) features computed after segmentation of nuclei were compared as well. The best destaining result was obtained by incubation of sections at 37 degrees C in preheated medium twice for 20 min. On average, the 3-D feature values were comparable with those after initial staining. With the described protocol it is possible to remove TO-PRO-3 fluorescence from tissue sections that can successfully be restained with minimal influence on fluorescence intensity and nuclear chromatin distribution.     

Human specific anti-type IV collagen monoclonal antibodies,characterization and immunohistochemical application

Summary This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

EGFR gene amplification in glioblastomas. Is there a relationship with morphology of tumor cell nuclei and proliferative activity?

OBJECTIVE: To confirm a relationship between histomorphology of glioblastomas and amplification of the gene for the epidermal growth factor receptor (EGFR) as the most important molecular biologic alteration in these tumors. STUDY DESIGN: In paraffin sections of surgical specimens from 71 primary resected glioblastomas, tumor cell nuclei in the region with the highest proliferative activity (Ki-67 immunostaining) were investigated morphometrically. Shape variables (roundness factor, Fourier amplitudes) and nuclear area were measured. Additionally, the numerical density of Ki-67-positive tumor cell nuclei was estimated. Differential polymerase chain reaction (PCR) was performed from paraffin sections of the same tumor area. The signals for the EGFR gene and IFN gamma reference gene were quantified densitometrically. RESULTS: Cases with distinct EGFR gene amplification (EGFR/IFN ratios > 5) revealed significantly lower mean values for several Fourier amplitudes, indicating a more regular nuclear shape when compared with cases without evidence of EGFR gene amplification (EGFR/IFN-ratios < or = 1). The Ki-67 index and nuclear area showed no significant differences between these groups. Although a large variation in nuclear morphology was observed for cases without evidence of EGFR gene amplification, discriminant analysis based on morphometric variables provided a good separation of these cases from cases with distinct EGFR gene amplification, with a high percentage of statistically correct reclassified cases. CONCLUSION: Our results provide evidence of a relationship between genetic alterations and histomorphology of glioblastomas.     

Nuclear localization of melatonin in different mammalian tissues: Immunocytochemical and radioimmunoassay evidence

Melatonin was detected by an improved immunocytochemical technique in the cell nuclei of most tissues studied including several brain areas, pineal gland, Harderian gland, gut, liver, kidney, and spleen from rodents and primates. Cryostat sections from tissues fixed in Bouin's fluid, formalin, or acetone/ethanol were used. The nuclear staining appeared primarily associated with the chromatin. The nucleoli did not exhibit a positive reaction. The melatonin antiserum was used in the range of 1:500 to 1:5,000. Incubation of the antibody with an excess of melatonin resulted in the complete blockade of nuclear staining. Pretreatment of the sections with proteinase K (200–1,000 ng/ml) prevented the positive immunoreaction. In a second aspect of the study, we estimated the concentration of melatonin by means of radioimmunoassay in the nuclear fraction of several tissues including cerebral cortex, liver, and gut. The subcutaneous injection of melatonin (500 μg/kg) to rats resulted, after 30 min, in a rapid increase in the nuclear concentration of immunoreactive melatonin which varied in a tissue-dependent manner. However, samples collected 3 h after the injection showed that melatonin levels had decreased to control values. Pinealectomy in rats resulted in a clear reduction in the nuclear content of melatonin in the cerebral cortex and liver but not in the gut. The results of these studies suggest that melatonin may interact with nuclear proteins and that the indole may have an important function at the nuclear level in a variety of mammalian tissues.     

Immunofluorescent Staining of Leptospires in Pepsin Treated Histologic Sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

Immunofluorescent staining of leptospires in pepsin treated histologic sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

Colorieetric Tests of Nuclear Staining

The materials of these investigations were the mucosal nuclei of formalin-paraffin sections (stained by hematoxylin and eosin) from the human uterus in carcinoma, non-malignant hyperplasia and in health. By a scale made of water color pigments placed near the magnified images of nuclei projected on a screen, it was demonstrated mathematically that, for a given specimen, nuclear staining intensity varies inversely with nuclear volume. The most probable explanation is the greater separation of chromatic particles in the larger nuclei.     

Colorieetric Tests of Nuclear Staining

The materials of these investigations were the mucosal nuclei of formalin-paraffin sections (stained by hematoxylin and eosin) from the human uterus in carcinoma, non-malignant hyperplasia and in health. By a scale made of water color pigments placed near the magnified images of nuclei projected on a screen, it was demonstrated mathematically that, for a given specimen, nuclear staining intensity varies inversely with nuclear volume. The most probable explanation is the greater separation of chromatic particles in the larger nuclei.     

Basic Fuchsin and Picro-Indigo Carmine: A Selective Stain for Cells in Mitosis and for Autoradiography

Selective staining of dividing nuclei is accomplished as follows: paraffin sections, after hydration, are stained 15 min in a saturated aqueous solution of basic fuchsin, washed, then stained 1.5 min in an equal-volumes mixture of indigo carmine saturated in 70% alcohol, and saturated aqueous picric acid. Removal of excess dye with 3 changes of 70% alcohol, dehydration, clearing and covering in a resinous medium completes the process. Nuclei of dividing cells are stained red; cytoplasm and interphase nuclei, light green. This method has been used successfully for determining the mitotic activity of skin, kidney, liver and other rabbit and mouse tissues. Tissue sections previously prepared as autoradiographs may be stained by this method to facilitate the determination of radioactive labeling of mitotic cells.