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PubMed1994

Metal chelates as reversible stains for detection of electroblotted proteins: application to protein microsequencing and immunoblotting.

Coomassie brilliant blue and Ponceau red have traditionally been used to stain electroblotted proteins, since they are compatible with existing N-terminal and internal protein microsequencing as well as with immunoblotting procedures. With recent improvements in sequencing and immunoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alternatives to conventional stains. Electroblotted proteins were detected by blocking nonspecific sites with polyvinylpyrrolidone-40 followed by incubation in metal chelate solutions at acidic pH values. Two of the most promising metal chelate stains were the Ferrozine/ferrous complex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electroblots were successfully stained using iron chelates. When these two stains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stains was substantiated by incubating stained membranes at neutral to basic pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapidly elute the complexes from the bound proteins. The chelate stains were determined to be fully compatible with immunoblotting, N-terminal, and in situ internal protein microsequencing.

Amino Acid Sequence↗

PubMed1975

The interaction of apurinic acid aldehyde groups with pararosaniline in the Feulgen-Schiff and related staining procedures.

The equilibrium reactions involved in the formation of the apurinic acid (APA)-Schiff chromophores in the staining phase of the Feulgen-Schiff reaction do not allow a quantitative conversion of APA to these chromophores. By modification of the sulfite and dye concentrations and the pH of the staining reagents, or by using better solvents for pararosaniline like acetic acid or dimethylsulfoxide (DMSO) a shift of these equilibria was attempted in order to obtain a higher amount of APA-bound dye. A 40% higher absorbance, when compared with the normal Schiff-staining, was obtained in model films by staining with a saturated solution of pararosaniline in a 1:1 v/v mixture of DMSO and SO2-water, followed by rinsing in SO2-water. A doubling of the absorbance resulted in the same objects when a saturated solution of pararosaniline in a 2 M acetic acid/acetate buffer of pH 4.45 was used for staining, followed by a short rinse in SO2-water. Amino groups (as found in histones) are shown to compete with the amino groups of pararosaniline for the APA aldehydes. This effect, although causing lower staining intensities, is shown not to be the explanation for the differences in stain content found between more and less compact forms of chromatin. Depending on the pH, and dye and sulfite concentrations of the staining reagents, the following components are considered as possible contributors to the mixture of chromophores (Duijndam et al., 1973 b) formed between APA and Schiff's reagent or its modifications: 1. An acid labile component with a wavelength of maximal absorbance (lambda max) near 510 nm; its structure is probably the azomethine--CH=N--; 2. A relatively acid stable component with a high value of molecular absorbance (epsilon), an lambda max near 570 nm and possibly having an enamine structure--CH=CH--NH--; 3. A component with intermediate acid stability, low epsilon, and lambda max near 540 nm, and which is probably an alkylsulfonic acid --CH(SO3H)--NH--compound. Small differences in the staining conditions in the histochemical application of the Feulgen-Schiff reaction may cause a shift in the ratio between especially components 2 and 3, resulting in variations in stain content and in lambda max.

Acetates↗

PubMed1995

Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique.

The well-known technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects, our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue in the microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.

Animals↗

PubMed1976

Further observations on the chemistry of pararosaniline-Feulgen staining.

Pararosaniline-Feulgen staining of cells in suspension produces nucleus- and chromatin-specific fluorescence as well as color. Experiments were designed to test postulated reaction mechanisms responsible for the fluorescent staining with the nonfluorescent pararosaniline. The reduction in fluorescent-staining intensity by pretreatment of cells with 2.2 x 10-2M K2S2O5 tends to rule out the alkysulfonic acid pathway; conditions favoring the formation of this intermediate reduce staining intensity. The fluorescence enhancement, observed when cells stained in pararosaniline without K2S2O5 are post-treated with K2S2O5, suggests that there is an initial Schiff-base linkage between pararosaniline and an aldehyde of hydrolyzed DNA, and that this linkage is stabilized in the presence of K2S2O5. Microspectrofluorometer measurements of cells stained at various pararosaniline concentrations in 2.2x10-2M K2S2O5, show that the fluorescence emission maximum ranges from about 627 nm at 3.1x10-3 M pararosaniline to about 604 nm at 3.1x10-5M. All of the employed staining protocols appear to produce the same fluorescent product, perhaps a heterocyclic pyronin analog formed from pararosaniline. Flow microfluorometric analysis of cells stained in suspension verified that the relative fluorescence intensity represents relative DNA content. Staining at reduced pararosaniline concentration (3.1x10-4M) reduces the coefficient of variation of the flow microfluorometric histograms, showing that maximum quantitation does not necessarily correlate with maximum staining intensity.

Animals↗

PubMed1986

Cell blotting: techniques for staining and microscopical examination of cells blotted on nitrocellulose paper.

Standard cytological and histological staining procedures were successfully applied on nitrocellulose (NC) paper for staining a spectrum of cell types that were blotted on it. The staining procedures included a Papanicolaou method, a hematoxylin and eosin method, and an immunoperoxidase technique. The cell types consisted of various suspension and monolayer cultures as well as cells freshly prepared from tumor and nontumor tissues. Suspended cells were manually blotted on NC paper; they were fixed in one of the fixatives, which included 80% 2-propanol, 10% neutral-buffered formalin, Carnoy's solution, Bouin's fluid, and B-5 fixative; they were then stained by one or more of the above methods. After staining, the NC paper was dehydrated in absolute 2-propanol; made transparent by soaking in xylene; mounted permanently in a mounting medium (Permount) on a glass slide; and then examined under a microscope. The protein-blotting capacity of NC paper (Schleicher & Schuell) was compared with those of the conventional cell blotting media (Millipore and Gelman) which contained cellulose acetate. Bovine serum albumin was dot-blotted on the NC and the cellulose acetate filters, and stained for protein by amido black. As in cytological/histological staining, the amido black-stained filters were dehydrated in absolute 2-propanol, made transparent in xylene, and mounted in Permount. Densitometric tracings indicated that the NC paper bound the highest amount of protein. The feasibility of cytological/histological staining techniques on NC paper combined with its high protein-blotting capacity allowed in situ staining and microscopical characterization of baby hamster kidney cells binding to fibronectin blotted on the NC paper. The possible significance of these techniques in current cell biological research was discussed.

Animals↗

PubMed1986

The use of a simple haematoxylin and eosin staining procedure to demonstrate micronuclei within rodent bone marrow.

In the pharmaceutical industry, the majority of drug-safety evaluation studies are carried out preferentially in the rat. Consequently, drug absorption, distribution, metabolism and excretion profiles are available for this species. Such data usually have to be generated independently in the mouse, to allow validation of any micronucleus tests carried out in this species. Unfortunately, at the present time, the rat is not ideal for use in the micronucleus test due to the presence of large numbers of contaminating mast cell granules. Such granules are stained blue by the most commonly accepted staining procedure (May-Grunwald-Giemsa), and can be erroneously scored as micronuclei when they overlay erythrocytes. A simple haematoxylin and eosin staining procedure was evaluated in the micronucleus test using rats and mice. With this procedure, micronuclei stained blue-black and were readily distinguishable from cell inclusions resembling micronuclei such as mast cell granules, which remained unstained. Essentially similar quantitative data for micronucleus incidence and erythrocyte distribution were obtained in mice using this staining technique when compared to the use of the more established May-Grunwald-Giemsa staining procedure. However, unlike the use of the May-Grunwald-Giemsa procedure, the use of the haematoxylin and eosin stains allowed the accurate estimation of micronucleus incidence within the marrows of treated rats in the presence of contaminating mast-cell granules. Furthermore, unlike alternative procedures using fluorescent stains, the haematoxylin and eosin stained preparations are stable, constitute a permanent record of the experiment, and can be analysed at the convenience of the investigator. Therefore, this staining procedure may offer a useful alternative, for example, when evaluating rat bone-marrow smears within which considerable mast cell contamination can occur.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1984

Diffraction patterns from stained and unstained helices: consistency or contradiction?

Addressing the issue of how best to integrate negative staining electron microscopy with X-ray fiber diffraction of macromolecular helices, we investigated staining properties of helical models using computational simulations. We compared diffraction patterns of stained and unstained representations of F-actin, myosin S1-decorated actin, and microtubules, to test the reliability of selecting well preserved specimens as those whose optical diffraction patterns most closely match the X-ray patterns of hydrated fibers. We conclude that if the stain layer is considerably thicker than the outer diameter of the specimen, this criterion is likely to be reliable. However, specimens in such thick stain layers may be resolution-limited by dynamic scattering effects such as beam-broadening. With relatively thin staining (the more practically relevant situation) "edge effects" in the stain distribution result in differences between diffraction patterns of stained and unstained specimens. Diffraction pattern changes due to molecular distortions such as shrinkage or flattening were similarly modeled. Since differences between diffraction patterns of stained and unstained helices may be due to either "edge effects" or molecular distortions, it does not appear possible a priori to distinguish between these effects. Comparison of experimental data for the (2.3 nm)-1 layer-line of tobacco mosaic virus (as model system) reveals major differences between the X-ray diffraction pattern of hydrated sols and the Fourier transform of HREM images of negatively stained specimens.

Microscopy, Electron↗

PubMedDate not supplied

Computer image analysis of two-dimensional crystals of beef heart NADH: ubiquinone oxidoreductase fragments. I. Comparison of crystal structures in various negative stains.

We investigated the structure of two-dimensional crystals from bovine heart mitochondrial NADH: ubiquinone oxidoreductase. A detailed description of uranyl acetate-stained crystals demonstrated that they are composed of fragments in a spatial arrangement according to space group P4212 [J. Brink, S. Hovmöller, C.I. Ragan, M.W.J. Cleeter, E.J. Boekema and E.F.J. van Bruggen, European J. Biochem. 166 (1987) 287]. To gain more structural information on the crystal structure and to assess the effects of various negative stains on the structure preservation and appearance, we examined stained crystals by means of electron microscopy and image analysis. The space group P4212 appeared to be present for several stains tested, i.e. ammonium molybdate, uranyl acetate, uranyl nitrate and uranyl sulphate. Use of phosphotungstic acid and silicotungstate resulted in a reduction of symmetry to pseudo-P4212 or p4. Use of sodium tungstate led to a considerable loss of resolution to 3.8 nm at best, whereas otherwise 1.5 to 1.9 nm could be demonstrated. The lattice vectors were not affected by the stains; they were determined as a = b = 14.9 +/- 0.25 nm with gamma = 89.8 degrees +/- 0.6 degrees. Image analysis showed the presence of similar structures with the molybdate and uranyl compounds. Differences were observed in the case of the tungstate type of stains. Furthermore, the analysis revealed the complete absence of the four small pores of 2.0 nm diameter in the unit cell. This effect was observed irrespective of the type of stain and supporting film, and could be ascribed only to the glow-discharge treatment of the supporting film. The observed difference must be caused by changed interactions between the protein, stain and supporting film. Application of correspondence analysis and clustering algorithms to the various reconstructed images of the crystals showed that they could be separated into several clusters. Each of these clusters corresponded on the average to only one type of stain, whereas a further division according to the specific uranyl compounds was observed. This study therefore shows that under identical preparation conditions subtle differences between individual stains can be detected.

Animals↗

PubMed1990

Ethanol-phosphotungstic acid and bismuth staining of spermatid nucleoli in mouse spermiogenesis.

The nucleoli of developing mouse spermatids were examined with ethanol-phosphotungstic acid (E-PTA) staining, and also with bismuth staining following formaldehyde fixation (FA-Bi staining) and glutaraldehyde fixation (GA-Bi staining). Only the cortical zone of the nucleolar dense fibrillar component (DFC) in the round spermatids was stained with E-PTA, while the inner area remained either faintly (early Golgi-phase spermatids) or completely unstained (cap-phase spermatids). Incubation of the fixed testis with dithiothreitol before E-PTA staining resulted in homogeneously intense staining of the DFC. The facts suggest that numerous E-PTA-positive basic proteins were present in the DFC, but disulfide crosslinks formed in the DFC proteins prevent penetration of PTA into the DFC interior. The DFC was stained with bismuth after FA-Bi and GA-Bi staining until the disappearance of the nucleoli occurring in acrosome-phase spermatids. The fibrillar center, homogeneously stained using E-PTA, FA-Bi, and GA-Bi methods was present in the nucleoli of Golgi-phase and early cap-phase spermatids, but disappeared in the nucleoli of late cap-phase spermatids. These results are discussed based on the previous studies dealing with the ribosomal RNA synthesis in mouse spermiogenesis.

Animals↗

PubMed2004

Fluoro-Jade, but not Fluoro-Jade B, stains non-degenerating cells in brain and retina of embryonic and neonatal rats.

Fluoro-Jade (FJ) and Fluoro-Jade B (FJB) are fluorescein derivatives currently used to stain brain cells under degeneration. In this study, we investigated the FJ staining of nondegenerating cells in embryonic and neonatal rat brain and retina. In embryonic rat brain (embryonic day 15; E15), very intense staining of cells was observed. The number of FJ-stained cells and the intensity of staining decreased with increasing in animal age, being almost absent by postnatal day 16 (P16). Only a few cells in neonatal rat brain were in the process of cell death, as verified by the TUNEL technique. The FJ-stained cells in neonatal brain were positive for the neuronal marker neuronal nuclei antigen (NeuN). In retina, FJ stained mainly cells from the ganglion cell layer at P2 and the neuroblastic layer at P2 and P6. In contrast to FJ, FJB did not stain nondegenerating cells in embryonic and neonatal rats. These results show that in addition to staining degenerating brain cells, FJ also stains nondegenerating central nervous system cells in embryonic and neonatal stages.

Animals↗

PubMed2004

Klippel-Trénaunay syndrome: the importance of "geographic stains" in identifying lymphatic disease and risk of complications.

BACKGROUND: Klippel-Trénaunay syndrome (KTS) is a rare congenital anomaly classically defined as the triad of vascular stain, soft tissue and/or bony hypertrophy, and venous varicosities. OBJECTIVE: To determine whether the morphologic characteristics of the associated vascular stains in KTS are predictive of the presence of lymphatic involvement and/or complications. SETTING: Outpatient dermatology practice, tertiary care medical center. METHODS: We retrospectively reviewed all cases of KTS identified between January 1989 and September 2001 at the University of California San Francisco (UCSF) Department of Dermatology. Forty patients were identified. We further classified them by type of cutaneous vascular stain, either "geographic" or "blotchy/segmental." Patients were further classified as having definite, probable, possible, or no evidence of lymphatic disease. We also reviewed the charts for other possibly associated manifestations and complications of KTS. RESULTS: Of those with sharply demarcated geographic stains (n=22), 21 had definite or probable evidence of lymphatic disease. Of those with blotchy port-wine stains (n=17), 16 had possible or no evidence of lymphatic disease (P <.001). Determination of the type of stain had 95% sensitivity and 94% specificity in differentiating the definite or probable presence of definite or probable lymphatic disease from possible or no evidence of lymphatic disease. Complications occurred in 19 (86%) of 22 patients with a geographic stain vs 7 of 17 (41%) with a blotchy/segmental stain (P <.003). CONCLUSION: This study demonstrates that the presence of a geographic vascular stain is a predictor of the risk of both associated lymphatic malformation and complications in patients with KTS. Since these stains are present at birth, this clinical observation can help in identifying individuals with KTS at greatest risk for complications and in need of closer observation.

Cellulitis↗

PubMed2005

A modified technique for high-resolution staining of myelin.

This report describes a new modification of the Gallyas method for staining myelin in fixed brain tissue and compares results of multiple myelin-visualizing techniques in normal common marmoset (Callithrix jacchus), normal macaque monkey (Macaca mulatta), and a human with multiple sclerosis. The new modification involves immersion in 10% formalin following impregnation in ammoniacal silver nitrate, and the use of a low concentration of 4% paraformaldehyde in the developer. This improved technique is less sensitive to post-mortem tissue handling, temperature, and minor contaminants, allowing a more straightforward implementation in the laboratory setting. It permits simple user-controlled development of the reaction product to maximize contrast in the area of interest, resulting in high contrast staining not only of large axonal bundles, but also thin fascicles throughout tissue sections. Myelin staining in visual cortex of an Old World monkey and a New World monkey reveals similar patterns in the new myelin silver stain, the Gallyas stain, and myelin basic protein immunohistochemistry. The most heavily myelinated areas occupy the edges of blobs, but neither the most lightly stained nor the most darkly stained areas are always in our outside a blob. This indicates a more complex pattern between myelinated axons and blobs than previously suggested. While the new myelin silver stain, darkfield microscopy, the Luxol Fast Blue stain, the Gallyas stain, and myelin basic protein immunohistochemistry all permit visualization of myelin in the CNS, each technique has its own merits and pitfalls; careful evaluation of individual study requirements would best determine which methods are the most useful.

Animals↗

PubMed2007

Measurements of DNA damage on silver stained comets using free Internet software.

Silver stain offers the possibility to stain comets permanently, but up to now it was impossible to measure the majority of the comet parameters, because the distinction between head and tail was not recognised by software. Here, we report a silver staining protocol that allows the measurement of comet parameters using the free Internet software CASP. We validated the silver stain protocol by comparing the behaviour of the parameter '% DNA in tail' in silver and fluorescent stained comets. The range of % DNA in tail for different visual categories of damage in silver stained comets was similar to that reported with fluorescence staining. The range was for category 0 (no damage), <1%; category 1 (low damage), 1-25%; category 2 (medium damage), >25-45%; category 3 (high damage), >45-70%; category 4 (very high damage), >70%. The mean of % DNA in tail in silver stained comets was also similar to that reported with fluorescence staining. The mean was for category 0, 0.4+/-0.34%; category 1, 12+/-7%; category 2, 37+/-4%; category 3, 57+/-5% and category 4, 83+/-6%. Others comet parameters such as tail length, tail moment and Olive tail moment can be also measured. The silver staining protocol reported here opens new opportunities for those working in the assay without fluorescent microscope as the measurement of comet parameters using free Internet software and conventional microscope becomes possible.

Comet Assay↗

PubMed2001

Methylene blue staining: is it really useful in Barrett's esophagus?

BACKGROUND: If areas of specialized intestinal metaplasia (SIM) or dysplasia in Barrett's esophagus can be identified at endoscopy, the number of biopsies could be reduced and the sensitivity of biopsy surveillance would increase. It has been suggested that methylene blue (MB) dye staining may be useful for this purpose. METHODS: Nine patients were prospectively studied with Barrett's esophagus. Staining involved sequential spraying of 10% N-acetylcysteine, 0.5% MB and water. Quadrantic biopsies were obtained from Barrett's epithelium and collected in separate containers depending on whether they were taken from stained or unstained areas. Seven patients undergoing yearly surveillance were asked to compare the discomfort of this endoscopy with that of previous surveillance endoscopies. Biopsies were analyzed for the presence and the percentage of SIM and dysplasia by a nonblinded pathologist. RESULTS: MB staining prolonged endoscopy by a mean of 8 minutes (47% increase in procedure time) and was associated with significant vomiting during the procedure in 2 patients. Staining was observed in all 9 patients. All 7 patients undergoing yearly endoscopic surveillance indicated more discomfort with endoscopy plus MB staining. Of 37 biopsies from stained mucosa, 20 contained SIM; of 23 from unstained mucosa, 15 contained SIM (57% sensitivity, 32% specificity for MB staining). CONCLUSIONS: In this small, nonblinded study MB staining was associated with prolongation of endoscopy, increased patient discomfort, and potentially serious complications and was neither very sensitive nor specific for SIM. It is our recommendation that this technique should not be routinely used in endoscopic surveillance of patients with Barrett's esophagus. Further studies of MB staining are needed.

Barrett Esophagus↗

PubMed1983

In vivo staining test with methylene blue for bladder cancer.

An in vivo staining test with 0.2 per cent methylene blue was applied to 129 patients with bladder tumor and 16 patients with chronic cystitis within a 6-year interval. Although normal mucosa did not pick up the stain nonpapillary in situ and microinvasive carcinomas did so frequently. Moderate dysplasia was stained in about half of the patients. The intensity of the stain in papillary tumors was correlated with the histologic anaplasia (grade). Grade 1 tumors were stained poorly or unstained in 86 per cent of the tests, whereas grades 2 and 3 tumors picked up the stain in 74 and 96 per cent of the tests, respectively. Even a tiny tumor, if poorly differentiated, was identified easily by the blue stain. The histologic anaplasia of tumors could be assessed roughly according to the intensity of the stain. However, chronic cystitis occasionally took up the stain, especially in cases of marked inflammatory infiltrate a deep stain was recognized. To differentiate nonpapillary early cancer from chronic cystitis the addition of a cytologic examination may be necessary.

Biopsy↗

PubMed1989

Application of Grimelius argyrophil staining to the study of tumour ultrastructure. III. Effects of osmium tetroxide.

The results obtained in electron microscopy for Grimelius (1968) argyrophil staining by the en bloc staining of Vassallo and the staining on section of Håkanson were compared on tumoral and non-tumoral tissue samples and were found to lead to different results. Our earlier results for the en bloc staining method indicated that the fixative used in this process was important and that embedding in resin followed by staining of section weakened silver impregnation. We examined here the effects of OsO4 post-fixation combined with such embedding and found that staining was obtained within a shorter period and was more intense. This staining on section was not specific enough, since it was observed in secretory granules of non-argyrophil cells such as those containing somatostatin. In addition, this staining was not confined to specific peripheral zone as it occurred after en bloc staining; particularly in secretory granules with double structure including a core such as those containing glucagon in the pancreas or certain granules in tumoral carcinoid tissue. Consequently silver deposits were located both in the core and peripheral zone of such granules. Moreover, after OsO4 post-fixation, embedding in resin, and staining on section, argentaffin and argyrophil properties intermingle.

Fixatives↗

PubMed1998

A simple and sensitive method for the activity staining of xanthine oxidase.

Xanthine oxidase is a commercially-important enzyme. Several biochemical compounds have been quantitated by xanthine oxidase. Xanthine oxidase has been used as an auxiliary enzyme in the staining of several enzymes or tissues, however, there is no direct staining method available for it, on polyacrylamide gels. Partially-purified xanthine oxidase from cow milk was used as the enzyme source for the development of an activity-staining method on polyacrylamide gels. Staining was very sensitive. Detection of 0.02 microU of the enzyme on polyacrylamide gels was possible. Staining of 0.05 microU takes about 1 min whereas staining of 0.5 microU will take less than 5 s. Addition of TEMED is not essential for activity staining but it did increase both the rate and the intensity of the staining. The stained gels must be washed with distilled water, extensively, in order to remove excess unoxidized nitroblue tetrazolium, and must be protected from light, for a clear background and sharp activity-band staining. This method might be useful for quality control of xanthine oxidase obtained from different sources.

Animals↗

PubMed2003

A robust, efficient and flexible method for staining myelinated axons in blocks of brain tissue.

Previous studies have demonstrated the utility of the gold chloride method for en bloc staining of a bisected brain in mice and rats. The present study explores several variations in the method, assesses its reliability, and extends the limits of its application. We conclude that the method is very efficient, highly robust, sufficiently accurate for most purposes, and adaptable to many morphometric measures. We obtained acceptable staining of commissures in every brain, despite a wide variety of fixation methods. One-half could be stained 24 h after the brain was extracted and the other half could be stained months later. When staining failed because of an exhausted solution, the brain could be stained successfully in fresh solution. Relatively small changes were found in the sizes of commissures several weeks after initial fixation or staining. A half brain stained to reveal the mid-sagittal section could then be sectioned coronally and stained again in either gold chloride for myelin or cresyl violet for Nissl substance. Uncertainty, arising from pixelation of digitized images was far less than errors arising from human judgments about the histological limits of major commissures. Useful data for morphometric analysis were obtained by scanning the surface of a gold chloride stained block of brain with an inexpensive flatbed scanner.

Animals↗