A Method for Silver Impregnation of Mammary Myoepithelia

Histochemical methods for microscopic visualization of nummary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

Appraisals and pitfalls of myoepithelial cell staining by Levanol Fast Cyanine 5RN.

In the search for specific staining of myoepithelial cells in human normal and pathologic mammary tissue we applied the milling dye Levanol (Supranol) Fast Cyanine 5RN. Reports from literature on this staining method were considered, and efforts were made to improve the procedure. Different fixatives were tested, and the pretreatment of the sections was modified. Five nuclear stains were tried but were finally omitted. Counterstain of the connective tissue proved useful. The advantages of the different modifications are discussed.     

Appraisals and pitfalls of myoepithelial cell staining by Levanol Fast Cyanine 5RN

Summary In the search for specific staining of myoepithelial cells in human normal and pathologic mammary tissue we applied the milling dye Levanol (Supranol) Fast Cyanine 5RN. Reports from literature on this staining method were considered, and efforts were made to improve the procedure. Different fixatives were tested, and the pretreatment of the sections was modified. Five nuclear stains were tried but were finally omitted. Counterstain of the connective tissue proved useful. The advantages of the different modifications are discussed.     

Application of silver stains to gastric endocrine cells in plastic sections

Summary A simultaneous light and electron microscopic study of mouse gastric mucosa was made to determine whether the silver nitrate methenamine stain of Duk-Ho Lee could be used to stain gastric endocrine-like cells in plastic embedded tissue. Examination of consecutive thick and thin sections showed that this stain blackened the granules of the predominant type of endocrine-like cell present. Blackening of the granules with silver occured in tissue fixed in osmium tetroxide solution with or without dichromate salt or in tissue fixed in glutaraldehyde then treated with osmium. The intensity of staining was deepest in the osmium-dichromate fixed tissue, but the glutaraldehyde-osmium procedure gave less interference from diffuse silver impregnation and better preservation of detail for electron microscopy.     

Human placenta: de visu demonstration of interstitial Cajal-like cells

Traditional interstitial cells of Cajal (ICC) are present in the digestive tube and are supposed to act as pacemakers and neuromodulators. However, interstitial Cajal-like cells (ICLCs) were found outside the gastrointestinal tract, in various organs (e.g. ureter, bladder, fallopian tube, uterus, pancreas, mammary gland, myocardium etc.) and looking for such ICLC is a priority in our laboratories.We report here unequivocal visual evidence that ICLCs are present in the mesenchymal tissue of the villi from human term placenta.The following methods were used: a. vital staining with methylene blue (cryosections); b. silver impregnation (paraffin sections); c. Epon-embedded sections (approximately 1 microm) of glutaraldehyde/osmium fixed tissue, stained with toluidine blue; d. primary cell cultures (or second-passage cells) to reveal the characteristic, very long, moniliform cell processes and mitochondrial localization at dilations (molecular fluorescence probe: Mito Tracker Green); e. immunofluorescence for c-kit/CD117 marker or other characteristic proteins; f. transmission electron microscopy to establish the identity of ICLC.     

Postembedding Staining of Brain Tissue for Ultrastructural Visualization of Amyloid in Alzheimer's Disease

A postembedding staining method is presented for ultrastructural visualization of amyloid deposits in brain sections from patients with Alzheimer's disease. Methenamine silver stain is applied to thin sections of tissue embedded in the acrylic resin LR Gold. Senile plaques are easily labeled by silver granules and the ultrastructural detail is well preserved. When staining time is prolonged, silver precipitate also is deposited on neuronal paired helical filaments. This method overcomes the drawbacks of previously reported applications of the stain on Vibra-tome and Epon sections. Thin sections from the same tissue block can be immunostained with antibodies to various plaque components, thus allowing comparative studies at the electron microscope level.     

A Modified Silver Impregnation Technique for the Observation of Thick Sections of Lymph Nodes Perfused with Colloidal Carbon

For many years, a variant of the silver impregnation technique of Bielchowsky has been used to study the lymph node because it clearly outlines the various structures which are usually hard to contrast with standard staining methods. Like other variants of silver impregnation, this method blackens the cell nuclei as well as the reticular fibers; however, it inhibits the impregnation of the nuclear chromatin immediately adjacent to fibers. Hence, this variant selectively darkens the lymphoid cell populations of the nodal structures which contain a loose fiber network.

To study the blood vascular network of the lymph node based on perfusion of colloidal carbon, a staining procedure was needed which would contrast nodal structures on thick sections, while allowing the carbon-filled small blood vessels to be distinguished from the impregnated coarse reticular fibers. In an attempt to adapt this variant of Bielchowsky's technique, 10, 20, 40 and 60 nm thick sections from rat nodes, fixed in a solution of Bouin-Hollande for 72 hr, were silver impregnated with serial dilutions (1:2 to 1:128) of the ammoniacal silver solution. Forty-micrometer thick sections impregnated with a 1:16 dilution of the original silver solution at 37 C and for 30 min provided the best results for the conditions.     

A silver carbonate method for cell counts of neurons and glial elements on paraffin embedded brain tissue.

A modification of the Del Rio-Hortega method for the demonstration of central nervous system elements is presented. This silver impregnation technique is particularly useful for the classification of cell types for quantitative differential cell counts. Formalin fixed paraffin sections are immersed in formol-ammonium bromide for 1 1/2 hours; this solution is an excellent mordant for various silver nitrate stains. The samples are stained for 20 to 60 minutes in a silver carbonate solution (25 ml of 25% silver nitrate combined with 200 ml of 5% sodium carbonate) and then reduced in a 1% formaldehyde solution to which 20 drops of acetic acid have been added. Finally, the slides are fixed in sodium thiosulfate, rinsed in tap water, dehydrated, cleared, and mounted. This procedure will enable this investigator to identify neurons, oligodendroglia, and astrocytes on the basis of their nuclear staining as well as to demonstrate the laminae of brain tissue since the method allows differentiation of cell layers and fiber tracts.     

Differential expression of human tissue factor in normal mammary epithelial cells and in carcinomas.

BACKGROUND: Tissue factor (TF) is a glycoprotein which binds factor VIIa. The TF-VIIa complex serves as a potent initiator of the coagulation pathways. TF, an immediate early gene, may also play a role in cell growth. Expression of TF was correlated with some types of cancers. MATERIALS AND METHODS: Normal, immortalized, and tumor human mammary epithelial cells were used in the experiments. The differential display (DD) technique was used to identify genes differentially expressed in the cells. TF expression patterns were examined by Northern blot analysis, immunofluorescence staining of cultured cells, and immunohistochemical staining in human cryostat sections. RESULTS: In a 5-way display, an amplified polymerase chain reaction (PCR) product was found in normal and immortalized human mammary epithelial cells but not in the breast cancer cells. The PCR fragment was cloned and sequenced. The result showed that the fragment was identical to human tissue factor. Northern blot analysis showed that expression level of tissue factor mRNA remained high in growing, quiescent, and senescent normal mammary epithelial cells. Immunofluorescence staining also confirmed tissue factor expression pattern in the cell lines tested. Immunohistochemical staining showed that tissue factor was expressed in the normal luminal and myoepithelial cells of some ducts but not others. No staining was observed in invasive carcinoma cells. However, myoepithelial cell staining was seen in some residual ductal structures in invasive tumors. CONCLUSIONS: This study shows the use of DD to reveal the loss of TF expression pattern in human breast cancer cell lines. Immunohistochemical staining results showed breast carcinoma cells expressed little TF, if any, suggesting that TF is not required for breast tumor cell invasion. The results also indicated that TF expression was independent of the proliferation status of the expressing cells. The expression pattern of TF may be a meaningful marker in the development of breast cancer.     

A modification of the tannic acid-phosphomolybdic acid-dye stain for demonstrating myoepithelial cells in formalin fixed tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin sections are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:1 methanol:acetic acid, rinsed in 9:1 methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

A simple impregnation technique for thin and semithin enterochromaffin cells in sections

Summary Constant, intense and precise impregnation of enterochromaffin (EC) cells was achieved simply by floating thin or semithin sections of gut mucosa, fixed in osmium tetroxide or in glutaraldehyde with postfixation in osmium, on a silver nitrate or proteinate solution. EC cells alone showed impregnation in the light microscope. In the electron microscope, impregnation affected not only the secretory granules of EC cells but also, although much more faintly, those of other, non-EC cells (D, X, D₁, G and other cells). Lysosomes also showed partial or total reactivity. Oxidation reduced but did not entirely suppress EC cell staining and had no effect on non-EC endocrine cell staining. Since the reaction did not occur with glutaraldehyde alone, osmium appeared to be a crucial component of the process. These findings should be borne in mind in applying Thiery's method for vicinal glycol groups to the type of study material used in these experiments.     

A simple impregnation technique for thin and semithin enterochromaffin cells in sections

Constant, intense and precise impregnation of enterochromaffin (EC) cells was achieved simply by floating thin or semithin sections of gut mucosa, fixed in osmium tetroxide or in glutaraldehyde with postfixation in osmium, on a silver nitrate or proteinate solution. EC cells alone showed impregnation in the light microscope. In the electron microscope, impregnation affected not only the secretory granules of EC cells but also, although much more faintly, those of other, non-EC cells (D, X, D1, G and other cells). Lysosomes also showed partial or total reactivity. Oxidation reduced but did not entirely suppress EC cell staining and had no effect on non-EC endocrine cell staining. Since the reaction did not occur with glutaraldehyde alone, osmium appeared to be a crucial component of the process. These findings should be borne in mind in applying Thiery's method for vicinal glycol groups to the type of study material used in these experiments.     

Effect of Temperature on Argyrophil Impregnation: Development of A High Temperature Rapid Argyrophil Procedure

Out studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

Effect of temperature on argyrophil impregnation: development of a high temperature rapid argyrophil procedure

Our studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin

Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contraction, Acta2 null mice were utilized and milk ejection and myoepithelial cell contractile force generation were evaluated. Pups suckling on Acta2 null dams had a significant reduction in weight gain starting immediately postbirth. Cross-fostering demonstrated the lactation defect is with the Acta2 null dams. Carmine alum whole mounts and conventional histology revealed no underlying structural defects in Acta2 null mammary glands that could account for the lactation defect. In addition, myoepithelial cell formation and organization appeared normal in Acta2 null lactating mammary glands as evaluated using an Acta2 promoter-GFP transgene or phalloidin staining to visualize myoepithelial cells. However, mammary myoepithelial cell contraction in response to oxytocin was significantly reduced in isolated Acta2 null lactating mammary glands and in in vivo studies using Acta2 null lactating dams. These results demonstrate that lack of ACTA2 expression impairs mammary myoepithelial cell contraction and milk ejection and suggests that ACTA2 expression in mammary myoepithelial cells has the functional consequence of enhancing contractile force generation required for milk ejection.     

Modification of the Silver Proteinate Impregnation Technique for Protozoa and Cultured Nerve Cells

Silver impregnation with silver-protein compounds is widely used for staining tissue sections and cell cultures. Some authors report that the results obtained with these methods have not always been reproducible because the reagent's composition varies according to the manufacturer. To avoid this problem in the method described in this paper, a silver proteinate, produced in our own laboratory is used. Although our method is based on Bodian's, the modifications we have made allows its use for both free-living cells (protozoa) and cells grown in culture (nerve cells). The significant modifications are 1) different fixation, 2) postfixation with Cajal's for-mol-bromide, 3) changes in the duration of the impregnation steps technique and 4) elimination of metallic copper. The method reported here enables us to use silver proteinate whenever we require it and to control the composition of the silver proteinate. This technique can be used for cells cultured in either plastic or glass.     

Immunocytochemical identification of proliferating cell types in mouse mammary gland

To study cell proliferation in different cell types and segments of the mammary gland, we devised a dual staining procedure, combining nuclear labeling by 5-bromo-2'-deoxy-uridine (BrdU) uptake (revealed by a dark-brown precipitate) and an alternative (red or blue) cytoplasmic labeling by antibodies specific for the differentiation proteins of epithelial, myoepithelial, and secretory cell types. The following markers, revealed by APAAP or beta-galactosidase procedure, were selected: alpha-smooth muscle actin for the myoepithelial cells, keratin (detected by AE1 monoclonal) for the luminal epithelial cells, alpha-lactalbumin and beta-casein for the secretory cells. To follow the full process of organogenesis, the study was conducted in mouse mammary glands from virgin, primed, and lactating animals and from glands cultured in vitro under specific hormone stimulation. Cell proliferation was localized mainly in focal areas (end buds), and mostly corresponded to "null" undifferentiated cells. Estrogen and progestin stimulation induced a relative increase of proliferating differentiated cells of either epithelial or myoepithelial type, localized in ducts and alveolar structures. Prolactin stimulation induced proliferation in secretory cells.     

Ultrastructure of coelomic lining in echinoderm podia: significance for concepts in the evolution of muscle and peritoneal cells

Summary Ultrastructural data are presented on the histological organization of coelomic lining in the podia of ten species of the five major groups of extant echinoderms. Further evidence of the incorporation of podial retractor muscle cells (myocytes) into a monociliated myoepithelial coelomic lining is provided. In the podia of the crinoid Nemaster rubinginosa and the ophiuroid Ophiophragmus wurdemani as well as in the feeding tentacles of the holothurian Leptosynapta tenuis, coelomic linings are organized as simple myoepithelia consisting of non-contractile peritoneal cells (peritoneocytes) and myocytes. Coelomic linings in the holothurian Thyonella gemmata, the echinoids Eucidaris cf. tribuloides and Lytechinus variegatus, and the asteroids Asterias forbesi and Astropecten sp. are pseudostratified or bipartite pseudostratified myoepithelia consisting of subapical myocytes and apically situated peritoneocytes. The ophiuroid podia of Ophioderma brevispinum and Ophiothrix angulata exhibit transitions from simple myoepithelia to partially pseudostratified epithelia. Intermediate forms between the extremes in myoepithelial organization also occur in the podial lining of single species (e.g. Eucidaris cf. tribuloides). These data supplement recent ultrastructural studies on the podial lining of echinoderms and, in conjunction with published ultrastructural data on the myoepithelial organization of other coelomic linings in echinoderms and in other coelomates, suggest myoepithelial organization of the coelomic lining is a plesiomorph feature in Bilateria.     

A Modification of the Tannic Acid Phosphomolybdic Acid-Dye Stain for Demonstrating Myoepithelial Cells in Formalin Fixed Tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin section are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:l methanol:acetic acid, rinsed in 9:l methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

Suppression of Connective Tissue Impregnation in a Silver Technique for Demonstrating Nerve Fibers

A tissue pretreatment technique is introduced which effectively suppresses the silver impregnation of connective tissue and nompecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcobol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginnins with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilidng fresh hrozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation H suppressed by the use of mercuric chloride in the fixative and that the background supprgsion is related to the short fixation time with formol-sublimate.