Improved correction of quantitative nuclear DNA (ploidy) measurements in tissue sections

OBJECTIVE: To evaluate, using a computer model, the advantages for ploidy measurements of selecting only center-containing sections of nuclei in ultrathin, very thick or relatively thick tissue sections. STUDY DESIGN: The computed corpuscle sectioning program was run on a personal computer. Its synthetic data were corrected by a variety of correction algorithms. RESULTS: When only center-containing sections of nuclei were selected in ultrathin sections, spherical nuclei could be corrected perfectly, and mildly prolate ellipsoidal nuclei with a selection bias in favor of elliptical nuclear section profiles could be corrected with high fidelity. Ultrathin sections most faithfully represented the true height of the peak of highest ploidy and showed better peak discrimination than other choices of section thickness, but small sample size, wavy sections, markedly inhomogeneous intranuclear DNA distribution and oblate ellipsoidal nuclei represented significant limitations of this approach. As nuclear prolation increased, peak definition worsened, and the peak of highest ploidy was falsely shortened. Results were unaffected by errors in the estimation of section thickness when an internal diploid standard was used. The effect of variable internuclear DNA concentration in mildly or moderately prolate ellipsoidal nuclei was nil. The choice of correction algorithm was unimportant, except that the reference curve method was better able to analyze oblate ellipsoidal nuclei, wavy sections and nuclei with inhomogeneous intranuclear DNA, and provided superior insight into nuclear and section parameters. Thick and very thick sections did not require correction and, unlike ultrathin sections, were immune to markedly inhomogeneous intranuclear DNA distribution, to nonspherical nuclear shape and to focal variation in section thickness (waviness), but (in relatively thick more than in very thick sections) the height of the peak of highest ploidy was falsely shortened, often markedly, and peak definition was worse. CONCLUSION: Choice of section thickness and selection of only center-containing nuclear sections for analysis with a bias in favor of elliptical nuclear section profiles in ultrathin sections are very important for optimal results; the choice of correction algorithm is less important.     

Conceptual comparison of two computer models of corpuscle sectioning and of two algorithms for correction of ploidy measurements in tissue sections

OBJECTIVE: To compare two computer models of corpuscle sectioning and two algorithms for correction of ploidy measurements in tissue sections. STUDY DESIGN: Two models of corpuscle sectioning (the computed corpuscle sectioning program [CCSP] [Analyt Quant Cytol Histol 1997;19:376-386] and the ellipsoid sectioning program [ESP]) were run on a personal computer to generate synthetic corpuscle section data that model the sectioned nuclei in a tissue section. These synthetic data were analyzed by two algorithms for correction of ploidy measurements in tissue sections: the reference curve method (RCM) (Analyt Quant Cytol Histol 1997;19:376-386) and the method of McCready and Papadimitriou (MMP) (Analyt Quant Cytol 1983;5:117-123) for a variety of choices of section thickness and of nuclear section profile selection criteria. RESULTS: Previous recommendations (Analyt Quant Cytol Histol 1999;21:103-112) for optimization of ploidy analysis in tissue sections (selection of only center-containing sections of nuclei in ultrathin sections with a selection bias in favor of elliptical nuclear section profiles) are valid regardless of which corpuscle sectioning model and correction algorithm are employed. Perimeter correction may be desirable or necessary in some cases. The RCM has very significant advantages over the MMP, and the CCSP is more applicable to actual ploidy analysis than is the ESP. CONCLUSION: The RCM always should be used to correct ploidy measurements in tissue sections. The MMP should not be used as the sole method but, when used, should be used with and interpreted in the context of the RCM.     

Choice of methodology for quantifying cancer structures in tissue sections. A comparison of 2- and 3-dimensional estimators of mitotic activity, cellularity and nuclear size in breast cancer

OBJECTIVE: To analyze the relationships between a range of 2-(2D) and 3-dimensional (3D) estimators of important tumor features (mitotic activity, cellularity and nuclear size). STUDY DESIGN: Measurements were performed in systematically sampled fields of vision of 3-microns-thick sections and in optical disectors of 40-microns-thick sections of 93 breast cancers. RESULTS: 2D mitotic profile density and frequency correlate highly with 3D mitotic density and frequency (r > or = .75); the choice of estimator was of minor importance. 2D nuclear profile density correlated quite closely with 3D nuclear volume fraction and nuclear density (r > or = .66). Cellularity estimators were, however, influenced differently by nuclear size. 2D mean nuclear profile area and 3D volume- and number-weighted mean nuclear size correlated less closely (r > or = .51). Tumors with high mitotic counts generally had large nuclei, reflecting the fact that both variables are associated with aggressiveness. CONCLUSION: 2D and 3D quantitative estimates of corresponding tumor features often correlate closely. Easily applicable and unbiased 3D techniques are recommended for obtaining the mean size of nuclei (or other particles) and of volume fractions of structures. For ranking of cellularity and mitotic activity, 2D measurements are usually sufficiently accurate and close to corresponding 3D estimates. However, if exact quantities of tissue structures are required, unbiased (3D) techniques must be used.     

Combination of lamin immunocytochemistry and in situ hybridization for the analysis of chromosome copy numbers in tumor cell areas with high nuclear density

We describe the application of lamin immunocytochemistry (ICC) and single- or double-target fluorescence in situ hybridization (FISH) on 4 microm thick frozen tissue sections as a method to facilitate scoring of aberrant chromosome copy numbers in colonic tumors. Analysis of FISH signals in colon tissue sections is often hampered by overlap and truncation of epithelial nuclei, due to the density of the epithelial cells. Furthermore, on the basis of nuclear staining it is often difficult to determine whether or not nuclei are overlapping, or adjoining. Therefore, reliable evaluation of (F)ISH signals to screen for genomic changes was until now mainly restricted to isolated nuclei obtained from relatively thick tissue sections. In this study the applicability of lamin ICC, to stain the nuclear periphery and to distinguish individual nuclei, combined with the FISH procedure is explored to solve this problem for colon epithelium. For ICC we applied the alkaline phosphatase (APase)-Fast Red detection method, since the fluorescent precipitate of this reaction resists extensive proteolytic digestion as needed for efficient FISH on tissue sections. Chromosome copy numbers could easily be determined in 4 microm thick frozen tissue sections by combining lamin ICC and FISH. The ratio of the copy numbers of the chromosomes 7 and 17 could be determined in frozen tissue sections after combined lamin ICC and double-target FISH. It is concluded that the combination of lamin ICC and FISH improves chromosome copy number analysis and can be used to investigate genomic changes in different tumor compartments in thin frozen tissue sections.     

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.     

The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy

The nonchromatin structure or matrix of the nucleus has been studied using an improved fractionation in concert with resinless section electron microscopy. The resinless sections show the nucleus of the intact cell to be filled with a dense network or lattice composed of soluble proteins and chromatin in addition to the structural nuclear constituents. In the first fractionation step, soluble proteins are removed by extraction with Triton X-100, and the dense nuclear lattice largely disappears. Chromatin and nonchromatin nuclear fibers are now sharply imaged. Nuclear constituents are further separated into three well-defined, distinct protein fractions. Chromatin proteins are those that require intact DNA for their association with the nucleus and are released by 0.25 M ammonium sulfate after internucleosomal DNA is cut with DNAase I. The resulting structure retains most heterogeneous nuclear ribonucleoprotein (hnRNP) and is designated the RNP-containing nuclear matrix. The proteins of hnRNP are those associated with the nucleus only if RNA is intact. These are released when nuclear RNA is briefly digested with RNAase A. Ribonuclease digestion releases 97% of the hnRNA and its associated proteins. These proteins correspond to the hnRNP described by Pederson (Pederson, T., 1974, J. Mol. Biol., 83:163-184) and are distinct from the proteins that remain in the ribonucleoprotein (RNP)-depleted nuclear matrix. The RNP-depleted nuclear matrix is a core structure that retains lamins A and C, the intermediate filaments, and a unique set of nuclear matrix proteins (Fey, E. G., K. M. Wan, and S. Penman, 1984, J. Cell Biol. 98:1973-1984). This core had been previously designated the nuclear matrix-intermediate filament scaffold and its proteins are a third, distinct, and nonoverlapping subset of the nuclear nonhistone proteins. Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in matrix organization. Conventional Epon-embedded electron microscopy sections show comparatively little of the RNP-containing and RNP-depleted nuclear matrix structure. In contrast, resinless sections show matrix interior to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are covered with 20-30-nm electron dense particles which may contain the hnRNA. The large electron dense bodies, enmeshed in the interior matrix fibers, have the characteristic morphology of nucleoli. Treatment of the nuclear matrix with RNAase results in the aggregation of the interior fibers and the extensive loss of the 20-30-nm particles.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

ULTRASTRUCTURAL CYTOCHEMISTRY : Enzyme and Acid Hydrolysis of Nucleic Acids and Protein

Selective extraction of specific cell components by enzyme or acid hydrolysis is possible from ultrathin sections for electron microscopy and parallel 2 µ sections for light microscopy of tissues fixed in formalin and embedded in a water-soluble polyepoxide, product X133/2097. Normal rat tissues fixed 15 minutes in formalin at 3°C are more rapidly digested by proteinases than those fixed for the same length of time at 20°C. Trypsin selectively attacks the nuclear chromatin and the ribonucleoprotein particles of the ergastroplasm, whereas mitochondria and zymogen granules resist tryptic digestion. Pepsin rapidly attacks the mitochondria and zymogen granules. The ergastoplasm and nucleus at first resist peptic digestion, but in time the entire cytoplasm and interchromatinic portion of the nucleus are attacked. Ribonuclease abolishes cytoplasmic basophilia in 2 µ sections, but parallel ultra-thin sections, stained with uranyl acetate and examined in the electron microscope, show no change in the ribonucleoprotein particles of the ergastoplasm. Desoxyribonuclease alone had no effect, but after pretreatment of the sections with pepsin or hydrochloric acid, desoxyribonuclease specifically attacked the nuclear chromatin. Nucleic acid-containing structures in the sections are gradually disintegrated by perchloric acid or hydrochloric acid.     

Immunological analysis of the biochemical properties of the uterine estrogen receptor

Immunohistochemical and immunochemical analysis using Western blot techniques were carried out with estrogen receptor (ER) monoclonal antibody H-222 to 1) clarify the "nuclear translocation" phenomenon of ER, 2) elucidate the primary nuclear binding site of ER, and 3) to evaluate the binding force between ER and its nuclear binding site in the uterus of ovariectomized adult mice. Exclusive nuclear localization of ER was recognized in the epithelial cells, stroma cells, and smooth muscle cells. Uterine tissues prepared from animals injected with saline, 17 beta-estradiol (E2), estriol (E3), and diethylstilbestrol (DES) exhibited almost the same ER immunostaining when they were fixed prior to sectioning (prefixation method) and frozen sections were used. On the other hand, when fresh-frozen sections were fixed before or after incubation with various solutions (postfixation method) and then treated with various salt solutions, greater differences were seen in immunostaining of ER between saline-injected and hormone-treated animals. Immunostaining of ER in control animals was low after incubation with PBS (0.01 M phosphate buffer containing 0.16 M NaCl, pH 7.2), whereas uterine tissue from hormone-injected mice showed strong nuclear immunostaining after this treatment. After treatment with 0.4 M KCl or 0.5 M NaCl, immunostaining in the uterus of both hormone-injected and control animals was completely abolished. DNase treatment caused an almost complete loss of immunostaining of ER; however, RNase digestion slightly increased immunoreactivity in both E2-injected and control animals. Quantitative analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques showed that after incubation of tissue sections for 30 min with PBS, 0.4 M KCl, or DNase, 60%, 10%, and 30% of ER were present, respectively, compared to amount of ER present in unincubated sections. These findings suggest the following for the ER in uterine tissue; nuclear occupancy is a phenomenon that occurs due to a differential affinity between occupied and unoccupied receptors in the nucleus; after hormone treatment, the receptor levels do not fluctuate in the nucleus to the extent demonstrated by binding assays; and the properties of the ER detected in the immunohistochemical analysis are identical to those observed in biochemical studies.     

The use of glycol methacrylate embedding for studies of chromatin distribution and fine structure in salmon spermatid nuclei.

Glycol methacrylate has been used as an embedding medium for studies of spermiogenesis in the salmon. DNA and basic proteins are shown to stain with the same specificity in thick (0.5-1.0 mu) sections of GMA-embedded salmon testes as in sections of comparably fixed, paraffin-embedded testes. Stain can be localized far more precisely in GMA sections than in paraffin sections due to the thinness of the sections and to the excellent structural preservation of nuclei. In addition, ultra-thin sections of GMA-embedded salmon testes can be observed with the electron microscope, and this permits exact correlation between nuclear fine structure and chemical composition in consecutive sections of the same nuclei.     

Unusual structure of nuclei in a B–mutant strain of Schizophyllum commune with intercellular nuclear migration

Serial sections of multinucleate hyphae of a B–mutant strain of Schizophyllum commune , in which intercellular nuclear migration takes place, revealed appendages attached to nuclei. The appendages were strand–like organelles surrounded by a membrane with an electron–dense content. In addition, the outer membranes of the envelopes of adjacent nuclei were often joined and surface sections of these nuclei showed fibrous material with only a few nuclear pores. These ultrastructural features have not been reported before in the vegetative hyphae of S. commune , and they are suggested to be necessary for intercellular nuclear movement. The appendages may create the force or may result from the force necessary for the movement of the nucleus, while the joining of the outer membranes of the nuclear envelopes makes possible simultaneous movement of several nuclei, although only one has an appendage. The fibrous material in the nuclear envelope may facilitate sliding of the nuclei through the cytoplasm.     

Segmentation of transitional cell carcinoma nuclei by nonsupervised thresholding in different color spaces

OBJECTIVE: To develop a method for nonsupervised thresholding of transitional cell carcinoma nuclei of hematoxylin-eosin-stained histologic sections. STUDY DESIGN: For grayscale, RGB, HSL and L*a*b* thresholding we used an extension of a clustering method, based on a between-class/within-class criterion, applying optimal gray-level thresholding to distributions of R, G, B, or H and S or L* color domains. Algorithms were tested on 20 hematoxylin-eosin-stained sections of bladder carcinomas. RESULTS: Results were compared with corresponding results of manually selected nuclear areas. Images were compared pixel to pixel with matching reference images. Grayscale automatic thresholding presented unacceptably low pixel specificity, which complicated further nuclear segmentation. Nonsupervised thresholding in RGB or HSL, as well as semimanual thresholding in L*a*b* color space demonstrated significantly better accuracy and high values of pixel specificity and sensitivity, which permitted errors of only 4.27-5.83% in the subsequent mean area estimation of the transitional cell carcinoma nuclei. CONCLUSION: Nonsupervised multispectral thresholding in RGB or HSL color space extends single graylevel thresholding techniques to multilevel thresholding. This seems to be an effective, relatively simple and fast alternative to the widely used automatic grayscale or manual color thresholding for segmentation of nuclei in routine histologic sections.     

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.     

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.     

Iron and aluminum lakes of Gallo blue E as nuclear and metachromatic mucin stains.

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This coloring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast cells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent used and the amount of Al3+ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

Antigen retrieval by heating en bloc for pre-fixed frozen material.

Antigen retrieval (AR) is frequently required for successful immunohistochemistry (IHC) in archival formalin-fixed, paraffin-embedded tissue sections. Although AR by heating is most generally used, the majority of existing methods are useful only for paraffin-embedded sections. This article describes a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. The optimal temperatures for heating were 90C and above, and the optimal retrieval solutions were distilled water and 10 mM sodium citrate, pH 6.0. Sections were cut with a cryostat and mounted on poly-l-lysine-coated glass slides. After the sections dried, routine IHC was performed. Alternatively, free-floating sections were used. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. I examined the staining of 14 antibodies using sections of mouse brain and rat testis. The heating process was essential for five antibodies, improved immunoreactivity for seven antibodies, and provided no change for two antibodies.     

A bright field/fluorescent stain for aluminum: its specificity,validation, and staining characteristics

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.     

A bright field/fluorescent stain for aluminum: its specificity, validation, and staining characteristics

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.