Anti-native DNA antibodies from autoimmune sera also bind to DNA in mitochondria

Sera from 20 patients with systemic lupus erythematosus (SLE), selected for elevated titers of antibody to native DNA (nDNA), were examined by indirect immunofluorescence (IF) on tissue culture Hep-2 and rabbit kidney cells. Twelve sera showed a particulate cytoplasmic staining, in addition to nuclear IF. Double IF staining by using a mouse monoclonal anti-nDNA and a human serum containing anti-mitochondrial antibody as probes showed that the cytoplasmic structures recognized by these 12 SLE sera were mitochondria. SLE sera showing mitochondrial staining had high anti-nDNA levels, as assessed by ELISA (3.5 +/- 1.9 O.D.), compared with those not showing this staining pattern (0.8 +/- 0.4 O.D.). Mitochondrial staining was abolished by DNase I pretreatment of the substrates. Liquid phase absorption of serum anti-nDNA with S1 nuclease-treated calf thymus DNA or purified mitochondrial DNA also removed staining. These findings demonstrate that anti-nDNA antibodies from patients with SLE bind to DNA in intact mitochondria. Therefore, mitochondrial IF staining on tissue culture cells in the presence of nuclear staining should be interpreted with caution, because the phenomenon could be entirely related to anti-native DNA. These observations might also provide new insights concerning the nature of immunogenic cellular components stimulating anti-DNA production.     

Purification and characterization of an osteoclast membrane glycoprotein with homology to manganese superoxide dismutase.

The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the inorganic and organic components of bone matrix. Isolated avian osteoclasts have been used to immunize mice and generate an osteoclast-directed monoclonal antibody library (J. Cell Biology, 100:1592). A subset of these monoclonal antibodies recognizes antigens which are expressed on osteoclasts and which are absent or nearly so on multinucleated giant cells formed in vitro from monocyte or marrow mononuclear cells. One of these antibodies, designated 121F, has been used to identify and purify an osteoclast plasma membrane-associated glycoprotein. Western blot analysis on disulfide bond-reduced extracts from osteoclasts or multinucleated giant cells formed in vitro demonstrates that the 121F antibody recognizes a 150 kDa protein detectable only in osteoclasts. This high molecular weight protein has been purified by a combination of immunoaffinity and gel filtration chromatography procedures, in conjunction with electroelution of a single band from SDS-polyacrylamide gels. Silver staining of the purified antigen on SDS-polyacrylamide gels has revealed a single protein species larger than 200 kDa in its unreduced form and 150 kDa when disulfides are reduced. Isoelectric focusing of the purified antigen reveals a single species, having a neutral pl point of 6.95. Whereas endoglycosidase treatment and lectin affinity chromatographic analyses demonstrate that the antigen recognized by the 121F antibody possesses complex N-linked sugars, trifluoromethanesulfonic acid treatment indicates there are no additional O-linked carbohydrate components. Periodate oxidation and monosaccharide hapten inhibition studies provide no evidence for the antigenic epitope bound by the 121F antibody being carbohydrate in nature. Although the native antigen is blocked at its N-terminus, amino acid analysis of a hydroxylamine generated peptide disclosed a striking relationship between the osteoclast antigen recognized by the 121F monoclonal antibody and manganese and iron superoxide dismutase. Therefore, in addition to serving as a distinguishing cell type-specific marker for osteoclasts, this cell surface glycoprotein may function directly in osteoclast-mediated bone resorption.     

NF-kappa B sites function as positive regulators of expression of the translocated c-myc allele in Burkitt''s lymphoma.

An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.     

Plasma membrane and intracellular expression of globotetraosylceramide (globoside) in mouse bone marrow-derived mast cells

The cellular localization of globotetraosylceramide (globoside), one of the predominant neutral glycosphingolipids of mouse interleukin 3-dependent, bone marrow culture-derived mast cells (BMMC), has been determined by immunologic and chemical methods. Although less than 10% of BMMC expressed globoside on their surface, as assessed by cytofluorographic analysis of the binding of a mouse monoclonal IgM anti-globoside antibody, treatment of BMMC with nonactivating doses of pronase, trypsin, or neuraminidase increased the percentage of BMMC binding anti-globoside antibody by an average of six, three, or sixfold respectively. That most BMMC had globoside on their plasma membrane was confirmed by the surface radiolabeling of globoside with galactose oxidase and sodium borotritide, as detected by autoradiography of thin layer chromatograms of the extracted neutral glycosphingolipids. Thus, BMMC expressed globoside on their plasma membrane, but accessibility of a large probe such as IgM antibody to the glycosphingolipid was impeded by surrounding surface molecules. All BMMC bound anti-globoside antibody intracellularly, as assessed by indirect immunofluorescence staining and fluorescence microscopy on acetone-permeabilized cells, and the pattern of staining suggested that globoside was associated with the secretory granules of BMMC. Immunologic activation of BMMC resulted in a fivefold increase in the surface expression of globoside, as detected by cytofluorographic analysis of the binding of monoclonal anti-globoside antibody. The findings suggest that activation of BMMC causes a reorganization of the plasma membrane such that globoside is more exposed or that activation is accompanied by movement of globoside from internal membranes to the plasma membrane. The increased expression of globoside is a novel marker of the activated mouse BMMC.     

An immunocytochemical method for localization of estrogen receptors in rat tissues using a dinitrophenyl (DNP)-labeled rat monoclonal primary antibody.

We have developed an immunocytochemical method to demonstrate estrogen receptor in hormone-sensitive tissues of the rat using a dinitrophenyl (DNP) hapten-labeled rat antihuman estrogen receptor monoclonal antibody (MAb), H222. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase conjugate, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This method was applied to tissues from intact female rats and showed that estrogen receptor was localized in the nuclei of the stromal and glandular components of the uterine endometrium. Reduced receptor staining was observed in the luminal epithelium, with minimal myometrial staining. Anterior pituitary glands showed heterogeneous immunostaining and ovaries expressed the receptor predominantly in the interstitial cells; fallopian tubes demonstrated substantial epithelial staining. Uteri from chemically castrated rats showed reduced estrogen receptor immunostaining in both stromal and luminal cells, whereas staining was enhanced in the glandular elements. Classical estrogen-unresponsive tissues (heart, lung, and spleen) were unstained. Antibody controls involved pre-blocking antibody recognition sites on the receptor with unlabeled antibodies to estrogen receptor (H222, H226, and D547), as well as use of an inappropriate DNP-labeled antibody to metallothionein. These controls illustrated the specific nature of the DNP-H222 binding.     

Immunofluorescence in cells derived from Burkitt's lymphoma

Henle, Gertrude (Children's Hospital of Philadelphia, Philadelphia, Pa.), and Werner Henle. Immunofluorescence in cells derived from Burkitt's lymphoma. J. Bacteriol. 91:1248-1256. 1966.-Indirect immunofluorescence tests led to the brilliant staining of a small proportion of the cells in five different cultures derived from Burkitt's (African) lymphomas. The reaction was not restricted to the 17 sera from cases of this disease but extended to many sera from American individuals, whether healthy donors or patients suffering from a variety of illnesses. The incidence of positive sera increased with age from about 30% in childhood to > 90% in adults. Fluorescein-isothiocyanate-conjugated human gamma-globulins were suitable for direct staining of the same proportion of cells. The stained cells appeared to be in varying stages of degeneration, but cultural conditions leading to an increase in the cellular death rates failed to result in a rise in fluorescent cells. Several observations suggest that the stainable cells might be those which are seen to harbor virus particles under the electron microscope. Two cell lines derived from leukemic patients in this country also contained a small fraction of stainable cells but two others, and numerous primary human leukocyte cultures, gave consistently negative results. Attempts to relate the staining to known viral antigens have failed to implicate herpes simplex, varicella, cytomegalo, and reo viruses types 1, 2, and 3. The nature of the virus carried by the lymphoma cells as well as of the staining reactions remains to be determined.     

The electron microscopic immunohistochemistry of elastase-treated aorta and nuchal ligament of fetal and postnatal sheep

In conjunction with the immunoperoxidase and the immunoferritin methods, antielastin antibody was used to study the localization of elastin in untreated and elastase-treated elastic fibers of the nuchal ligament and the aorta of fetal and young adult sheep. In tissues not treated with elastase, the staining reaction for antielastin antibody was localized in the outer zones of the amorphous components and along the surfaces of the microfibrils ; the central zones of the amorphous components were unreactive. After mild elastase treatment, incompletely digested amorphous components showed staining both in their central and outer zones, and some of the microfibrils became unreactive. After extensive elastase treatment, small scattered amorphous components were still found in association with bundles of microfibrils. These components were stained diffusely by the antielastin antibody method but were not detectable by staining with uranyl acetate and lead citrate or with Kajikawa 's method for elastin; elastin was not detected on the surfaces of the microfibrils by any of the methods used. These findings were interpreted as indicating that the surfaces of the microfibrils are associated with small amounts of elastin, and that evenly stained amorphous components are composed of elastin, which is loosely arranged and allows the penetration of antielastin antibody. These observations support the concept that microfibrils serve an important role as a scaffold for elastin deposition in elastogenesis. Because of their high sensitivity, immunohistochemical methods for detecting elastin are useful to study partially degraded elastic fibers.     

Burkitt's lymphoma. Distinction of subgroups by morphometric analysis of the characteristics of 55 cell lines

In an attempt to clarify the controversy about the distinction between Burkitt's and non-Burkitt's small noncleaved lymphomas, 55 cell lines derived from 48 Burkitt's lymphoma patients were characterized by morphometry on plastic-embedded sections. The results of the measurements permitted the identification of five main cytologic types, with regard to nuclear size, nuclear area dispersion and irregularity of nuclear profiles. The presence of the Epstein-Barr virus (EBV) and the geographic origin of the tumors seemed to play essential roles in the determination of nuclear size, with a significantly larger size seen in EBV-positive cell lines, and especially in the African lines among these. Immunoglobulin profile and monoclonal antibody expression also correlated with the nuclear size. Two conclusions may be drawn from this analysis. An in vivo transformation of the cells of Burkitt's lymphoma can be postulated to explain the wide morphologic spectrum of lymphomas presenting a rearrangement of chromosome 8. The fact that typical and atypical Burkitt's lymphomas cannot be differentiated by study of their derived cell lines raises the question as to the validity of the distinction between the two subtypes of small noncleaved lymphomas.     

Cryptodeterminant of a sperm maturation antigen on the mouse flagellar surface.

The determinant of a mouse sperm maturation antigen was examined morphologically and biochemically with monoclonal antibody T21 as a probe. The plasma membrane components of cauda epididymal spermatozoa were extracted with nonionic detergent Nonidet P-40 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and by immunoblotting. Wheat germ agglutinin-lectin staining and immunoblotting indicated that the antigen recognized by T21 is a sialoglycoprotein of about 54,000 daltons (54 kDa). The antigenic determinant was more distinctly exposed after treatment with neuraminidase, as evaluated by immunohistochemistry, immunocytochemistry, and immunoblotting. The cryptic nature of the determinant was further confirmed by immunostaining nitrocellulose strips, subsequently digesting the strips with neuraminidase, and then reimmunostaining them. Results obtained by periodate oxidation treatment suggested that the epitope is a carbohydrate. Immunoperoxidase electron microscopy confirmed that the antigen is distributed on the flagellar plasma membrane of the sperm. This was demonstrated clearly when sperm were desialylated with neuraminidase. These results indicate that the 54 kDa sialoglycoprotein sperm maturation antigen has a cryptodeterminant which can be masked by a sialic acid residue, that is recognized by monoclonal antibody T21.     

COMPARATIVE STUDIES ON THE CELL WALLS OF SEXUAL AND ASEXUAL BANGIA ATROPURPUREA (RHODOPHYTA). I. HISTOCHEMISTRY OF POLYSACCHARIDES1

A comparative histochemical study of the nature and distribution of acidic and neutral cell wall polysaccharides was conducted on marine sexual and asexual filaments of the red alga Bangia atropurpurea (Roth) C. Ag. Outer and inner walls of this species were clearly partitioned according to staining and transmission electron microscopic characteristics. Neutral polysaccharides were detected in the outer coating (cuticle) but were absent from outer and inner walls of all filaments. Acidic polysaccharides were noted in the outer wall material but not in the inner wall layers of any filaments at any developmental stages. The major staining component of vegetative regions of sexual material and all regions of asexual filaments was a highly sulfated polymer. During sexual reproduction only there was a generalized change in the nature of the acidic component, characterized by a decrease in intensity of staining for sulfates in both male and female filaments and the appearance, in female filaments only, of polysaccharides which presumably were carboxylated. Spermatia attached to both male and female filaments in regions where sexual differentiation was initiated and where changes in the outer wall components commenced.     

Identification of the neutral glycosphingolipids of murine mast cells: expression of Forssman glycolipid by the serosal but not the bone marrow-derived subclass

The expression of neutral glycosphingolipids by mouse T cell-dependent, bone marrow-derived mast cells (BMMC) obtained in vitro was determined by chromatographic and immunochemical criteria. Neutral glycosphingolipids were isolated from BMMC by extraction of 3 to 5 X 10(8) cells in chloroform/methanol (1/1, v/v) and chromatography on DEAE-Sephadex, and were analyzed by thin layer chromatography with orcinol staining. The predominant neutral glycosphingolipids of BMMC were glucosylceramide (CMH), lactosylceramide (CDH), globotriosylceramide (CTH), globotetraosylceramide (globoside), and a molecule migrating slightly faster than gangliotetraosylceramide (asialo GM1) and slower than globopentaosylceramide (Forssman glycolipid). The profiles on thin layer chromatograms of the neutral glycosphingolipids were the same for BMMC derived from BALB/c, C57BL/6, WBBF1-W/Wv, and WBBF1-+/+ mice, and for cells differentiated in either WEHI-3 conditioned medium or concanavalin A-splenocyte conditioned medium. High performance liquid chromatography of benzoylated neutral glycosphingolipids of BMMC on a Zipax column confirmed the identity of the four neutral glycosphingolipids identified by thin layer chromatography. The fifth major glycosphingolipid had an elution time greater than that of globotetraosylceramide and did not co-elute with any of the standards tested. Direct biochemical analyses of the neutral glycosphingolipids of mouse serosal mast cells (SMC) were not feasible because only 2 X 10(6) SMC could be isolated per 100 mice. However, mouse SMC bound a rat monoclonal anti-globopentaosylceramide antibody (M1/87.27.7) and rat monoclonal B1.1 antibody, as assessed by indirect immunofluorescence and flow cytometry, whereas mouse BMMC did not. The binding of B1.1 antibody to SMC could be blocked by the anti-globopentaosylceramide antibody, and the specificity of B1.1 antibody for globopentaosylceramide was confirmed immunochemically with the use of a solid phase radioimmunoassay. As estimated immunochemically, the amount of globopentaosylceramide in mouse SMC was 62 ng/10(6) cells, whereas BMMC contained less than 8 ng/10(6) cells. Thus, the expression of globopentaosylceramide is a characteristic of the mouse SMC that is lacking in the T cell-dependent BMMC.     

Oncocytic carcinoma of the parotid gland. A case report

BACKGROUND: Oncocytic carcinoma is a rare malignant tumor of the salivary gland. Abundant, granular, eosinophilic cytoplasm is recognized as an oncocytic feature that reflects an accumulation of mitochondria. Ultrastructural study or immunohistochemical staining using antimitochondrial antibody can confirm the oncocytic nature of the tumor. However, there have been no data on whether immunocytochemical staining for human mitochondria aids in the confirmation of the oncocytic nature of oncocytic carcinoma. CASE: A 61-year-old man presented with a swelling in the left lower cheek. Computed tomography demonstrated a solid, isodense tumor in the parotid gland. An excisional biopsy of the tumor was performed, and an enlarged regional lymph node was removed. Imprint cytology of the lymph node showed cohesive cell clusters with lymphocytes. The clusters were composed of tumor cells that had characteristic abundant, granular cytoplasm and round to oval, centrally or eccentrically located nuclei with increased, fine chromatin and distinct nucleoli. Immunocytochemical staining revealed granular immunoreactivity of the cytoplasm for human mitochondria. Histology demonstrated tumor invasion in the normal gland and adjacent skeletal muscles. All tumor cells showed positive cytoplasm with antimitochondrial antibody by immunohistochemistry. Ultrastructural studies demonstrated packed mitochondria in the cytoplasm of the tumor. CONCLUSION: Immunocytochemical staining for human mitochondria help confirm the oncocytic nature of oncocytic carcinoma in cytologic specimens.     

Influence of cell fixatives and incubation schedule on PAP labeling of fibronectin in cultivated fibroblasts

A simple combined method for differential PAP labeling of fibronectin (FN) in mouse embryo fibroblast cultures was developed. Methanol-5% glacial acetic acid in dry ice-fixed cell monolayers showed mainly intracellular FN staining. Fixation with neutral paraformaldehyde before labeling, developed membrane- and extracellular matrix-associated FN. A combination of both procedures, which required incubation with primary antibody, fixation with paraformaldehyde followed by chilled acid methanol, and re-incubation with primary antibody, yielded sharp intracellular and extracellular FN labeling. The outlined methods can be readily employed in association with other staining techniques.     

Peripheral neurosecretory cells on the thoracic median nerves of the locust,Schistocerca gregaria

The distribution of neutral red staining peripheral cell bodies along the nerve trunks of the thoracic median nervous system of the locust, Schistocerca gregaria, is described. Backfilling of the cells with cobalt chloride solution reveals that they are neurones with characteristic axonal processes that terminate in the neurohaemal areas of the median nerve. The neurones react with the dye acridine orange, indicating their neurosecretory nature. This is confirmed by their ultrastructural appearance at the electron microscope level. The distribution and staining properties of the cells are compared with those of peripheral neurones from other insects and the nature of their neurosecretory product is discussed.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23α. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

A monoclonal antibody to animal centrosomes recognizes components of the basal-body root complex inChlamydomonas

Summary The mammalian centrosome monoclonal antibody MPM-13 recognized component(s) of the well defined MTOC basal-body root complex in the green plantChlamydomonas. The antibody reaction coincided in location with the basal-body root complex and the cruciate nature of the staining pattern corresponded to the configuration of the root microtubules. During mitosis the behaviour of MPM-13 stained material mirrored the duplication, separation and migration to the spindle poles of the basal body-root complex. It is suggested that conserved MTOC components were recognized and that these may have retained a similar, perhaps universal, function in microtubule organization.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidine-2-phenylindole dihydrochloride - mt mating type - MT microtubule - MTOC microtubule organizing centre - PFA paraformaldehyde - PBS phosphate buffered saline     

HISTOCHEMICAL STAINING AND CHARACTERIZATION OF GLYCOPROTEINS IN ACID-SECRETING CELLS OF FROG STOMACH

Glycoproteins were histochemically localized in oxyntic cells of the frog stomach by staining with periodic acid-silver methenamine. Reduction of silver was most intense on (a) the outer aspect of the apical plasmalemma, (b) within the tubular smooth membrane system characteristic of oxyntic cells, and (c) within cisternae and vesicles of the Golgi complex. Other membrane components such as those from the mitochondria, nucleus, junctional complex, lateral and basal cell membranes showed little or no stainability. Gastric mucosal homogenates were fractionated by centrifugation for further morphological and chemical analysis. The staining reaction of the microsomal fraction (40,000 g x 60 min) was similar to that of the tubular membranous components of intact oxyntic cells. Carbohydrate analyses showed that all cell fractions are extremely low in acidic sugars, uronic and sialic acids, while neutral sugars and hexosamines are relatively abundant. The microsomal fraction contains the largest proportion of carbohydrates, ca. 9% of the fat-free dry weight. Another distinguishing feature is that glucosamine is the only detectable hexosamine in the microsomal fraction. These histochemical and chemical data indicate that neutral glycoproteins are associated with membranous components which have been implicated in the process of HCl secretion by oxyntic cells. The staining pattern within the cells supports the hypothesis of interrelationships between the Golgi membranes, tubular smooth membranes, and apical surface membrane.     

Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells

A murine monoclonal antibody, termed HeFi-1, was produced after immunization with the L428 Hodgkin's disease tissue culture cell line. HeFi-1 selectively stained only the Reed-Sternberg or Hodgkin's cells in 18 of 18 cases of Hodgkin's disease, including the nodular sclerosis, mixed cellularity, and lymphocyte-depleted histologic subtypes. HeFi-1 did not stain any cells in normal lung, brain, salivary gland, thyroid, gall bladder, pancreas, liver, testis, breast, endometrium, or kidney. Rare large cells at the edge of the lymphoid follicles were stained in normal tonsil, colon, and hyperplastic thymus. There was no staining of any cells in 14 cases of B cell non-Hodgkin's lymphoma; however, the malignant cells in three of 11 cases of non-Hodgkin's lymphoma which appeared to express T cell markers were also stained with HeFi-1. Tissue culture cell lines including the T cell acute lymphocytic leukemia lines MOLT4 and CEM, the histiocytic cell line U-937, and the amniotic cell line WISH were not stained. Seven Epstein Barr virus (EBV)-positive lymphoblastoid cell lines were stained with HeFi-1, but there was no staining of three EBV+ African Burkitt's lymphoma cell lines or three EBV- American Burkitt's cell lines. HeFi-1 did not block the ability of the L428 cells to stimulate a mixed lymphocyte reaction or function as accessory cells for mitogen-induced human T cell proliferative responses. Modulation of the HeFi-1 cell surface antigen on the L428 cells was not observed. HeFi-1 specifically immunoprecipitated a cell surface protein of approximately 120,000 daltons from both the L428 and EBV+ lymphoblastoid cell lines. HeFi-1 monoclonal antibody should prove useful not only in the diagnosis, staging, and potential therapy of Hodgkin's disease, but also for determining the cell of origin of the Reed-Sternberg cell.     

Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry

Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry is more sensitive than other chromogens. Its instability in aqueous solutions and ethanol, however, has limited its application. We now report a method for stabilizing TMB by incubation in combinations of diaminobenzidine (DAB)/cobalt (Co2+)/H2O2. The stabilized TMB product was unaffected by long-term exposures to ethanol, neutral buffers, and subsequent immunohistochemical staining procedures. A procedure is recommended for optimal stabilization of TMB that affords a sensitivity for demonstrating retrogradely labeled perikarya comparable to standard TMB histochemistry. The physical characteristics of the reaction product make it suitable for combination with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunohistochemical staining procedure. This was established by staining retrogradely labeled neurons in the basal forebrain with a monoclonal antibody against choline acetyltransferase. Because the stabilized TMB product exhibited a superior sensitivity over cobalt ion intensification of the DAB-based reaction product (DAB-Co), it offers a distinct advantage over previously described combination procedures.     

Basic Fuchsin-Ferric Chloride: a Simplification of Weigert's Resorcin-Fuchsin Stain for Elastic Fibres

Weigert's resorcin-fuchsin stain for elastic fibres can be simplified by the omission of the resorcinol. The resulting “basic fuchsin-ferric chloride” gives results indistinguishable from those achieved with resorcin-fuchsin on tissues fixed in Bouin, Carnoy, Gendre, neutral formah, Susa or Zenker solutions. Gel filtration chroma tography on Sephadex LH20 showed that the staining components of basic fuchsin-ferric chloride had a high molecular weight, so selective staining of elastic fibres by this method may well be due to van der Waals attractions.