Fixation-dependent cytoplasmic false-positive staining with an immunoperoxidase method

Fixation-dependent nonspecific staining with the unlabeled immunoperoxidase (PAP) method was studied using paraffin-embedded human spleen sections fixed in various fixatives; the specific primary antiserum was omitted or nonimmunized normal rabbit serum was used. Strong cytoplasmic staining of polymorphonuclear leucocytes and macrophages was found after fixation in acetone, alcoholic formalin (94% alcohol) and absolute ethanol. This staining was mainly produced by the second layer of the PAP method. The most probable explanation of this phenomenon is nonspecific protein-immunoglobulin interaction as a result of alcoholic or acetone fixation of the sections. The present findings point to the importance of controls for each case under study to avoid false-positive interpretations.     

Adrenocorticotropin. 57. Synthesis and biological activity of ostrich and turkey hormones

Synthesis of ostrich and turkey corticotropin (ACTH) has been accomplished by the solid-phase method. Each was identical to the natural hormone in high performance liquid chromatography. Relative potencies in a lipolytic assay in isolated rabbit fat cells were: human ACTH, 100; ostrich ACTH, 53; turkey ACTH, 28. In isolated rat fat cells relative lipolytic potencies were: human ACTH, 100; ostrich ACTH, 2; turkey ACTH, 13. It was concluded that lipolytic potency is sensitive to alterations in structure throughout the entire length of the ACTH sequence in the rat fat cell assay.     

Fixation-dependent cytoplasmic false-positive staining with an immunoperoxidase method

Summary Fixation-dependent nonspecific staining with the unlabeled immunoperoxidase (PAP) method was studied using paraffin-embedded human spleen sections fixed in various fixatives; the specific primary antiserum was omitted or nonimmunized normal rabbit serum was used. Strong cytoplasmic staining of polymorphonuclear leucocytes and macrophages was found after fixation in acetone, alcoholic formalin (94% alcohol) and absolute ethanol. This staining was mainly produced by the second layer of the PAP method. The most probable explanation of this phenomenon is nonspecific protein-immunoglobulin interaction as a result of alcoholic or acetone fixation of the sections. The present findings point to the importance of controls for each case under study to avoid false-positive interpretations.Supported by the Sigrid Jusélius Foundation and Finska Läkaresällskapet     

The release of membrane-associated calcium from rabbit neutrophils by fixatives. Implications for the use of antimonate staining to localize calcium

Summary The addition of oxalate to a suspension of rabbit peritoneal neutrophils before fixation with glutaraldehyde and postfixation with osmium tetroxide-antimonate greatly enhanced the amount of calcium antimonate precipitate subsequently detectable with the electron microscope. Using chlortetracycline as a fluorescent probe for membrane-associated calcium, it was found that both glutaraldehyde and osmium tetroxide release calcium from membrane-associated stores in suspensions of living neutrophils. These findings suggest that some of the calcium released from cellular stores during fixation with glutaraldehyde is trapped within the neutrophil by oxalate which then reacts with potassium antimonate. This produces a more copious precipitate of calcium antimonate than fixation without oxalate. It is suggested, therefore, that the histochemical localization of calcium by antimonate techniques may not always represent thein vivo situation. The use of oxalate during fixation, however, may give a better indication of the amount of calcium stored within a cell.     

Pronase treatment increases the staining intensity of GABA-immunoreactive structures in the paravertebral sympathetic ganglia

Summary A novel tissue preparation technique for improving gamma-aminobutyric acid (GABA) immunocytochemistry has been developed. The influence of the glutaraldehyde concentration in the fixative and the effect of pronase treatment on the GABA immunostaining were tested. This method includes fixation with a high concentration of glutaraldehyde, gelatin embedding and treatment of the sections with pronase. In sympathetic (paravertebral) ganglia and their connectives, the most intense and specific immunoreaction was obtained with the following procedure: immersion fixation in 5% glutaraldehyde, infiltration and embedding in 15% gelatin, secondary fixation of the samples with 4% formaldehyde, floating frozen sections and digestion with 0.1% pronase for 15–20 min. With this technique, the GABA-containing structures (cells and nerve fibers with varicosities forming basket-like networks around some principal neurons) were selectively labeled. The data presented suggest that (1) a high concentration (5%) of glutaraldehyde in the primary fixative is necessary to preserve a large proportion of the GABA content; (2) this glutaraldehyde fixation partly masks the GABA immunoreactivity; and (3) this masking may be overcome by a proteolytic treatment preceding the immunostaining. This method has been extensively tested for the light microscopic visualization of GABA-containing tissue components in the sympathetic ganglion chain, but it may probably also be used for the immunocytochemical detection of other small molecules in other parts of the nervous system.     

Application of a rapid avidin--biotin--peroxidase complex (ABC) technique to the localization of pituitary hormones at the electron microscopic level

The new avidin--biotin--peroxidase complex (ABC) technique was applied to ultrathin sections of rat pituitary that were fixed with glutaraldehyde and embedded in Araldite 6005. The primary antisera dilutions that are normally applied for 24-48 hr with the peroxidase-antiperoxidase (PAP) complex technique were used. High background was observed with the ABC method when incubation times were 12-48 hr. Tests were then conducted with shorter incubation times. The staining intensity was measured with a densitometer. Detectable stain was seen after only 15 min in dilutions of 1:10,000 anti-bovine luteinizing hormone (bLH beta), 1:8000 anti-rat thyroid-stimulating hormone (rTSH beta), and 1:20,000 anti-25-39-adrenocorticotropic hormone (25-39ACTH). Optimal LH staining was seen after 30 min, whereas optimal staining for TSH or ACTH required 1 hr. Stain was detectable with a dilution of 1:4000 anti-human follicle-stimulating hormone (hFSH beta) after 30 min and was optimal after 4 hr. Prolonged incubation times with these dilutions decreased the staining intensity because a deposit of high background was produced that appeared as a filigreed network over the cells. When higher dilutions were tested with 2-hr incubation times, optimal staining was seen with 1:30,000 anti-bLH beta, 1:24,000 anti-rTSH beta, 1:30,000 anti-25-39ACTH, and 1:8000 anti-hFSH beta. These tests demonstrate the potential of the ABC method for the rapid detection of small amounts of specific and nonspecific antibodies that are bound to pituitary cells.     

Combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining

Summary A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step — Incubation of the tissue sections with the monoclonal primary antibodies; Second step — biotinylated anti-rat or anti-mouse IgG; Third step — monoclonal PAP complex; Fourth step — ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step — the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4-and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells. Stronger staining intensity was obtained for desmin-like immunoreactivity (LI) (within myofibroblasts of the lamina propria and muscle cells of the blood vessels)-and vimentin-LI (within Sertoli cells, myofibroblasts of the lamina propria and fibroblasts of the interstitium) of the human testis. The full combination (6 step-reaction) permits the detection of smallest quantities of an antigen in sections of different tissues using highly diluted primary antibodies. The technique represents a further alternative among the immunocytochemical amplification methods.     

Immunocytochemistry with monoclonal anti-immunoglobulin as a developing reagent: application to immunoperoxidase staining and radioimmunocytochemistry

The development of two monoclonal antibodies for use as second antibodies in immunocytochemistry is described. The antibodies are produced by mouse X mouse hybrid myelomas, and are both of the IgG type. The two antibodies, RB23 and ND13, were used to detect neurophysin by three-step peroxidase-antiperoxidase (PAP) immunostaining, and were "internally labeled" with 3H-lysine for the radioimmunocytochemical localization of neurophysin, substance P, and tyrosine hydroxylase using rabbit first antibodies. The binding sites of RB23 and ND13 on the rabbit IgG antibodies were determined by solid-phase radioimmunoassay, using allotype-specific rabbit serum to compete with RB23 and ND13. It was found that both RB23 and ND13 are directed against the B4 kappa-light-chain allotype. The immunocytochemical localization of adrenocorticotropic hormone and somatostatin with rabbit primary antibodies was not achieved with RB23 or ND13, and it is proposed that these antibodies are not of the B4 allotype. The findings demonstrate that monoclonal second antibodies can be useful general reagents for conventional immunocytochemistry as well as for radioimmunocytochemistry. Furthermore, allotype-specific monoclonal second antibodies may prove useful in the simultaneous immunohistochemical localization of more than one antigen in a given tissue section.     

Immunocytochemical effects of thyroxine stimulation on the adenohypophysis of dwarf (dw) mutant mice

The effects of dietary thyroxine on the immunoreactivity of cells in the pars distalis of the adenohypophysis in dwarf (dw/dw) mice were determined by ultrastructural immunocytochemistry. In nontreated dwarfs only adrenocorticotropic hormone (ACTH) cells and luteinizing hormone (LH) cells showed positive reactions to their respective antibodies, whereas no cells showed immunoreactivity to antibodies to growth hormone (GH), thyroid-stimulating hormone (TSH), or prolactin (Prl). In dwarfs supplemented postnatally with dietary thyroxine for 9 wks, the treatment failed to produced immunoreactive GH, TSH or Prl cells. However, LH cells became more prominent and fully developed, with denser concentrations of immunoreactive particles overlying the secretory granules than occurred in nontreated dwarfs. In thyroxine-treated dwarfs, ACTH cells were similar in ultrastructural features and immunoreactivity to those in nontreated dwarfs.     

Enhanced immunocytochemical staining sensitivity in formalin-fixed and paraffin-embedded material by simultaneous use of PAP and ABC

The simultaneous use of two immunoperoxidase (IP) methods; e.g. the PAP (Peroxidase-antiperoxidase) and ABC (Avidin-Biotin) to localize a single antigen enhances the sensitivity of polyclonal antibodies (FVIII/RAg and laminin) or monoclonal antibodies (MABs) (against human B-, T-cells and macrophages) in routinely formalin fixed and in paraffin embedded material. The increased sensitivity was not accompanied by loss of specificity. With antibodies against Factor VIII related antigen (FVIII/RAg) and laminin the increased staining intensity of blood vessels were demonstrated in experimental rat transplantation tumors. The similar enhancement of immunocytochemical reactions was observed with biopsies of human brain tumors, where the monoclonal antibodies against white blood cells were used. The staining results were of comparable intensity as in snapfrozen tissue or after special embedding procedure for immunocytochemical purposes acc. to Bolton and Mesnard formol sucrose gum, sucrose paraffin; FSGSP).     

Model systems for the immunolocalisation of cis,trans abscisic acid in plant tissues

To explore the feasibility of immunolocalisation of endogenous abscisic acid (ABA), model systems were developed for testing quantitatively the sensitivity of the second antibody peroxidase/antiperoxidse (PAP) method for immunolocalisation of ABA on plant tissues. Exogenous (±)ABA was fixed to carrot sections on glass slides or to homogenised pea cotyledon material on microtitre plates, either directly by carbodiimide fixation or by glutaraldehyde fixation of ABA-protein conjugates linked through the C1 carboxyl by 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride (EDC). Backgrounds were decreased by including 0.1% normal goat serum in the incubations, by including 0.1% Triton X-100 as a wetter, by including glycine in the rinses after EDC fixation and by using low-pH rinses after incubation with the primary antibody. Serum antibodies recognising the peptide bond between the protein and abscisic acid were removed by preincubating the serum with acetic acid conjugated to protein. Positives were only accepted when they could be eliminated by adding an excess of ABA-protein conjugate in the primary antiserum. By using a soluble peroxidase reaction product to facilitate quantitation, the limit of reliable exogenous ABA detection was found to be only of the order of 1 pmol. For the histochemical immunolocalisation of endogenous ABA, better antisera and lower backgrounds will be required.The efficiency of fixation of exogenous ABA was determined using [³H] or [¹⁴C]ABA. When aqueous EDC or di-isopropyl carbodiimide (IPC) were used the fixation efficiency was low (up to 5%), but much higher efficiencies (up to 80%) were obtained using IPC vapour with freeze-dried material. Similarly efficient fixation of endogenous ABA in pea cotyledon material, as determined by gas chromatography-mass spectrometry analysis, was obtained using the same technique. The PAP method failed to detect fixed endogenous ABA in pea cotyledons, even though the total tissue amounts present exceeded 1 pmol, evidence that not enough of the ABA was accessible to the antibody.Abbreviations ABA abscisic acid - ACE-ALP acetic acid-alkaline phosphatase - EDC 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride - GC-MS gas chromatographymass spectrometry - IgG Immunoglobulin G - HSA humanserum albumin - IPC dinsopropyl carbodiimide - LINK goat anti-rabbit IgG - OD optical density - PAP peroxidase/rabbit antiperoxidase complex     

Simultaneous demonstration of multiple antigens by indirect immunofluorescence or immunogold staining

Summary Available techniques for light and electron microscopical double immunocytochemical staining are all associated with certain problems. We have developed a novel multiple staining procedure, which allows use of antibodies of differing specificities, raised in the same species (e.g. rabbit). Its essential features include 1) saturation of antigenic epitopes on the first layer primary antiserum by second (fluorophor- or gold-) labelled anti-IgG antibodies and 2) denaturation of free anti-IgG binding sites by formaldehyde vapour treatment. Various combinations of gastrin, somatostatin, glucagon, ACTH, growth hormone and enkephalin/endorphin antibodies have been tested at the light and electron microscopical level and have been found to give highly reproducible double- and triple-staining results. The technique has also been evaluated by use of cytochemical paper models. The method is simple and very useful for multiple staining of a wide variety of antigens.     

Lysis of small cell carcinoma of the lung tumor cell lines by gamma interferon-activated allogeneic peripheral blood mononuclear cells: abrogation of killing by pretreatment of tumor cells with gamma interferon

Summary Interferon has been shown to enhance the ability of nonspecific cytotoxic mononuclear cells to lyse some, but not all, tumor cells. We have examined the effect of recombinant human gamma interferon (rIFN) on the cell-mediated cytolysis of tumor target cells derived from continuously cultured lines of small cell carcinoma of the lung (SCCL). Cells from the SCCL lines DMS 44, 53, 79, 92, and 406 were labeled with ⁵¹Cr and incubated with normal and rIFN-stimulated peripheral blood mononuclear cells for 18 h at 37 °C and tumor cell lysis estimated by measuring ⁵¹Cr release. Although cells from certain SCCL lines were good targets for cell mediated cytotoxicity, susceptibility to lysis was heterogenous among the different SCCL lines. DMS 406 and 79 were, on average, maximally lysed, while DMS 44, 53, and 92 showed less susceptibility to lysis by either control or rIFN-stimulated effector cells. In addition, although pretreatment with rIFN increased the cytolytic capacity of normal peripheral blood mononuclear cells from several different donors, preincubation of the tumor cell lines with rIFN resulted in inhibition of cytolysis mediated by both control and IFN-activated effector cells. These findings suggest that although rIFN may enhance cell-mediated lysis of SCCL tumor cells, it may also decrease susceptibility to lysis.This work was supported by Grants CA 37868, CA 31888, CA31918, CA33852, and AI 19053 awarded by the National Cancer Institute and Institute of Allergy and Infectious Diseases, DHHS. Statistical assistance was provided by Therese Stukel, biostatistician at the Norris Cotton Cancer Center, Hanover, H. H. and supported by Grant CA 23108 from the National Cancer Institute     

Simultaneous demonstration of multiple antigens by indirect immunofluorescence or immunogold staining. Novel light and electron microscopical double and triple staining method employing primary antibodies from the same species

Available techniques for light and electron microscopical double immunocytochemical staining are all associated with certain problems. We have developed a novel multiple staining procedure, which allows use of antibodies of differing specificities, raised in the same species (e.g. rabbit). Its essential features include 1) saturation of antigenic epitopes on the first layer primary antiserum by second (fluorophor- or gold-) labelled anti-IgG antibodies and 2) denaturation of free anti-IgG binding sites by formaldehyde vapour treatment. Various combinations of gastrin, somatostatin, glucagon, ACTH, growth hormone and enkephalin/endorphin antibodies have been tested at the light and electron microscopical level and have been found to give highly reproducible double- and triple-staining results. The technique has also been evaluated by use of cytochemical paper models. The method is simple and very useful for multiple staining of a wide variety of antigens.     

Human ferritin: effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.     

The effects of glutaraldehyde treatment and horesradish peroxidase conjugation on the immunoreactivity of bovine myelin encephalitogenic protein.

The effects of the immunoreactivity of bovine myelin encephalitogenic protein (EP) of treatment with glutaraldehyde and conjugation with horeradish peroxidase (HRP) were investigated by complement fixation and immunohistochemistry. Both glutaraldehyde treatment and HRP conjugation of EP decreased but did not abolish the reactivity of EP with rabbit anti-EP. Conjugation of EP to HRP by the two-step method of Avrameas had less detrimental effects on the immunochemical reactivity of EP than did the one-step procecure. The use of EP-HRP conjugates to probe for antibodies to EP is an immunochemically sound system with the major limitation that the number of antibody-producing cells is likely to be underestimated due to the failure to detect cells producing low levels of antibody or antibodies directed toward determinants altered by the modification procedures.     

Visualization of binding sites for bovine parathyroid hormone (PTH 1-84) on cultured kidney cells with a biotinyl-b-PTH (1-84) antagonist

Parathyroid hormone (PTH) receptors have been found in a subpopulation of kidney cells. In this report, we investigated the feasibility of techniques that apply a partial antagonist of PTH conjugated to biotin to localize receptors cytochemically on bovine kidney cortical cells in monolayer culture at the light microscopic level. Biotinylated bovine PTH (1-84) (biotinyl-PTH) was bound to the cultured cells for 1-30 min at 37 degrees C in the amounts of 10(-5) -10(-10) M. In a different set of experiments, the cells were also exposed to a solution containing 10(-6) M biotinylated PTH and an excess of unlabeled PTH, insulin, adrenocorticotropin, or calcitonin for 10 and 30 min at 37 degrees C to test the specificity of the binding. The cells were then fixed in 2.5% glutaraldehyde and stained with the avidin-biotin peroxidase complex (ABC) technique. Diffuse labeling was evident on 30% of the cells in 10 min with concentrations of biotinyl-PTH as low as 10(-8) M. The stain was diffuse, but more intense after 1-10 min in higher concentrations (10(-6) M). If a 15-1500-fold excess of unlabeled PTH was added to the biotinyl-PTH, no staining was observed. The other peptides (insulin, ACTH or calcitonin) had no effect on binding. Longer times in biotinyl-PTH (10(-6) M for 10-30 min) resulted in intense patches of label on the cells resembling caps (in addition to the pale diffuse label). The percentage of labeled cells in the monolayer (30%) did not change with time. These studies show that a partial antagonist of PTH can be used as a cytochemical probe for specific PTH receptors in a subpopulation of cultured cortical kidney cells.     

A cell strain cultured from porcine kidney increases cyclic AMP content upon exposure to calcitonin or vasopressin.

Cells originally dispersed from whole juvenile male Hampshire pig kidney and maintained in monolayer culture, increased cyclic AMP content in response to incubation with salmon calcitonin or antidiuretic hormone. Parathyroid hormone and epinephrine did not affect cyclic AMP content. The apparent K_m for arginine vasopressin in the porcine cells was 3.0 nM which is similar to the value obtained in single segments of rabbit kidney tubule. The apparent K_m for salmon calcitonin of 2.7 nM is higher than that reported for the rabbit nephron segments, but comparable to the K_m obtained in rat kidney homogenates. Exposure of the porcine cells to exogenous prostaglandin E₂ did not affect cyclic AMP responses to other hormones. In the cultured porcine kidney cells the pattern of hormone response is similar to that observed in nephron segments prepared from the medullary portion of the thick ascending limb of the loop of Henle, and these findings suggest that the porcine cells may be related to cells present in the medullary region of the kidney tubule.     

A serum-free medium supplemented with multiplication-stimulating activity (MSA) supports both proliferation and differentiation of chondrocytes in primary culture

The effect of hormones influencing cartilage metabolism on the growth of chondrocytes isolated from rabbit and rat ribs was investigated in serum-free medium. Insulin supported growth of the cells slightly, whereas calcitonin and parathyroid hormone did not. On the other hand, multiplication-stimulating activity (MSA), a substance partially purified from serum-free medium conditioned by the growth of Buffalo rat liver cells, markedly induced not only proliferation of the chondrocytes but also their synthesis of acid mucopolysaccharides, the characteristic cartilage phenotype, in serum-free medium. These cells maintained this specialized cellular function of differentiated chondrocytes for at least 21 days in serum-free medium. A combination of MSA and other hormones, such as insulin, calcitonin, and parathyroid hormone was even more effective in stimulating sulfation of glycosaminoglycans. These rabbit and rat chondrocytes cultured in completely defined medium seem to be a good experimental system for studies on chondrogenesis and endochondral ossification.     

Human ferritin: Effects of antigen source and fixation on leucocyte staining by immunoperoxidase technique

Summary The localization of ferritin was studied in peripheral blood cells and variously fixed tissues with the antibodies against ferritins isolated from human heart and spleen. The unlabelled antibody enzyme method (PAP) was used to detect the binding sites of antibodies. In peripheral blood cell smears both antisera gave rise to strong staining of polymorphonuclear (PMN) cell cytoplasm, whereas the monocytes stained relatively weakly. There were no staining differences between the two antisera. In human spleen sections the spleen ferritin antiserum stained the PMN cells and sinusoidal lining cells, whereas the heart ferritin antiserum stained only PMN cells. Neither of the two antisera stained monocytes in the spleen sections. This finding was observed in specimens fixed in Bouin's fixative, Baker's fixative and neutral formalin. However, the immunoreactivity of ferritin was totally destroyed by some other fixatives (Carnoy's fixative, formol sucrose and glutaraldehyde). These results suggest that ferritin is more readily released from monocytes than from PMN cells, and that mature spleen macrophages contain antigenic determinants of ferritin that are recognized only by anti-spleen ferritin antiserum.Supported by the Sigrid Jusélius Foundation and Finska Läkaresällskapet. Y.T.K. is a recipient of a grant for post-doctoral fellowship of Helsinki University