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.     

Human chromosome structure as revealed by an immunoperoxidase staining procedure

Morphological alterations accompanied by an increase of the glia-specific protein S-100 have been shown to occur in a glial cell line (138 MG) of a human brain tumour if serum is removed from the culture medium. The glial S-100 protein was immunologically indistinguishable from S-100 present in human brain.     

The influence of fixation on immunoperoxidase staining of plasmacells in paraffin sections of intestinal biopsy specimens

Summary The influence of different fixation methods on the results of immunoperoxydase staining of immunoglobulin and gastrin producing cells in gastric and duodenal mucosa was investigated. An indirect method was used on paraffin sections. It appeared that that fixatives containing sublimate gave the most consistent results, a sublimate-formaldehyde mixture being the best.     

The influence of fixation on immunoperoxidase staining of plasmacells in paraffin sections of intestinal biopsy specimens.

The influence of different fixation methods on the results of immunoperoxydase staining of immunoglobulin and gastrin producing cells in gastric and duodenal mucosa was investigated. An indirect method was used on paraffin sections. It appeared that that fixatives containing sublimate gave the most consistent results, a sublimate-formaldehyde mixture being the best.     

Rapid diagnosis of cytomegalovirus infections by direct immunoperoxidase staining with human monoclonal antibody against an immediate-early antigen.

Direct immunoperoxidase technique using a horseradish peroxidase (HRP)-conjugated Fab' fragment of human monoclonal antibody (humab C7), designated HRP-C7, was evaluated as a rapid diagnosis of cytomegalovirus (CMV) infection. A total of 138 clinical specimens consisting of 124 urine samples and 14 oral swabs were examined for CMV by the direct HRP-C7 staining in comparison with conventional virus isolation. The number of CMV-positive samples by each method was 40 (29.0%) for the former and 37 (26.8%) for the latter, respectively. By HRP-C7 staining, CMV was identifiable within 24 hr after inoculation. By conventional isolation method, an average of 10.3 days had passed before cytopathic effect characteristic of CMV appeared in the cell culture. Some false-positive and false-negative cases were discussed in relation to toxicity of urine samples, storage of the samples, and amount of CMV in the sample. The sensitivity and specificity of HRP-C7 method against conventional isolation method were 89.2% and 93.1%, respectively. Thus, HRP-C7 staining is useful for a rapid diagnosis of CMV infections.     

The effect of Tween 20 on indirect immunoperoxidase staining of blood group antigen A in human urothelium

The effect of the nonionic detergent Tween 20 on background staining, sensitivity, and specificity in the indirect immunoperoxidase staining for blood group antigen A was investigated histologically and spectrophotometrically. Pretreatment of dewaxed formalin-fixed Paraplast-embedded tissue sections from human ureters with 2% Tween 20 and dilution of the first and second layer antisera with 0.05 or 2% Tween 20 significantly reduced background staining of the urothelial cell cytoplasm, ureteral stroma, and musculature. Spectrophotometrical analysis of tissue sections from hypernephroma (rich in cytoplasm), cervix (fibrous stroma), and myometrium (musculature) underlined the histological results with a significant reduction of the maximum absorbance of Tween 20-modified indirect immunoperoxidase-stained tissue sections. Sensitivity, evaluated histologically by the endpoint titers of urothelial cell membrane staining, endothelial cell staining, and focal cytoplasmic staining of urothelial cells, was not influenced by the Tween 20 treatment. The specificity was improved as the staining was highly reduced or absent in control sections subjected to Tween 20.     

Sensitivity and nonspeicific staining of various immunoperoxidase techniques

Optimally fixed paraffin enbedded tissue sections and cytocentrifuged cell smears were used to test the sensitivity and nonspecific staining with the enzyme-bridge, PAP, indirect and direct immunoperoxidase methods using human immunoglobulins and lysozyme as antigens. With the enzyme-bridge method positive staining was seen with primary antiserum dilutions up to 1:20,000. The least background staining was observed with this method. The PAP method was equally sensitive, although false-negative results with low primary antiserum dilutions were seen. Some nonspecific background staining always persisted using the PAP method even with high primary antiserum dilutions. The indirect method was not as sensitive as the enzyme-bridge method and some nonspecific staining always persisted. The direct method was too insensitive with paraffin embedded tissue sections.     

Sensitivity and nonspecific staining of various immunoperoxidase techniques

Summary Optimally fixed paraffin embedded tissue sections and cytocentrifuged cell smears were used to test the sensitivity and nonspecific staining with the enzyme-bridge, PAP, indirect and direct immunoperoxidase methods using human immunoglobulins and lysozyme as antigens. With the enzyme-bridge method positive staining was seen with primary antiserum dilutions up to 1:20,000. The least background staining was observed with this method. The PAP method was equally sensitive, although false-negative results with low primary antiserum dilutions were seen. Some nonspecific background staining always persisted using the PAP method even with high primary antiserum dilutions. The indirect method was not as sensitive as the enzyme-bridge method and some nonspecific staining always persisted. The direct method was too insensitive with paraffin embedded tissue sections.Supported by the Sigrid Jusélius Foundation and Finska Läkaresällskapet     

Effects of fixation, dehydration and staining on dimensions of myxosporidan

The effects of fixation, dehydration and staining on the morphological dimensions of myxo- and microsporidan spores were tested. Seven fixatives, two dehydrants and five stains were tested. Ten % formalin produced the least shrinkage and provided the best cytological detail of fixed material in both types of spores. All fixatives caused shrinkage of myxosporidan spore length and polar capsule length. Spore capsule width and polar capsule width were unaffected by 10% formalin. Ethyl alcohol caused no significant change in spore width. Microsporidan spore length shrunk with all fixatives, but spore width was generally unaffected. Dehydration, with either isopropyl alcohol or acetone, produced additional, significant shrinkage. The influence of stains on spore size was negligible. Heidenhains iron hematoxylin followed by eosin, and Mallory's analine-blue collagen stain, effectively stained myxo- and microsporidan spores.     

Coral calcification rates by the buoyant weight technique: Effects of alizarin staining

The buoyant weight method has been used in a laboratory experiment over an 18-day period to assess effects of alizarin staining on the calcification rate of the hermatypic coral Diploria strigosa (Dana). Exposure of corals to a concentration of 10 mg/l alizarin for 24 h in a flow-through system caused a significant depression in calcification for a period of up to 6 days. It is suggested that such initial calcification depressions after staining could affect absolute growth measurements and cause synergistic effects with experimental manipulation of environmental conditions unless an appropriate recovery period is allowed.     

Fine needle aspiration diagnosis of malignant lymphoma with confirmation by immunoperoxidase staining

Cytospin preparations of fine needle aspirates in 14 cases of suspected lymphoma were studied by immunoperoxidase techniques. The combination of cytologic smears and immunoperoxidase studies resulted in a working diagnosis in 13 of the 14 cases. The immunologic markers in conjunction with the cytologic appearance of the aspirates were reliable and consistent in differentiating between malignant and benign lymphoproliferative lesions and in determining the B-cell or T-cell nature of the process.     

Visualization of peptide-immunoreactive processes on serotonin-immunoreactive cells using two-color immunoperoxidase staining

The sequential application of the peroxidase-antiperoxidase (PAP) technique with nickel-intensified DAB and with DAB alone was used to visualize black peptide-immunoreactive endings on amber serotonin-immunoreactive cells in 1-2-micron paraffin sections of the hamster medulla. Met-enkephalin- and substance P-positive terminals were present on serotonin cells in the raphe nuclei, the ventral reticular formation, and in the nucleus interfascicularis hypoglossi. The presence of enkephalin-immunoreactive endings on medullary serotonin-immunoreactive cells correlates with the analgesia and autonomic changes that result from the application of morphine or met-enkephalin to the medulla.     

Two-color immunoperoxidase staining: visualization of anatomic relationships between immunoreactive neural elements

A method for differentially staining for two antigens in single sections is described. Paraffin-embedded or Vibratome sections are incubated in two sequences of immunoperoxidase (PAP) reagents using a diaminobenzidine (DAB)-nickel ammonium sulfate solution to localize the first antigen, and DAB alone to localize the second antigen. With these chromagens, black and amber-colored reaction products are generated at the locations of the first and second antigens, respectively. The reaction products are stable and provide excellent color contrast. With this technique, the anatomical relationships between two sets of immunoreactive elements can be studied in individual sections. Intimate spatial associations that would probably not be detected by an examination of adjacent sections stained for each antigen can be visualized with this two-color immunoperoxidase method.     

X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining.

In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.     

LAK1: a novel leucocyte differentiation antigen shared by lymphoid and endothelial cells

Lymphokine activated killer (LAK) cells have been utilized as a useful tool in cancer adoptive immunotherapy. The lineage origin of this population has always been controversial since it shares phenotypic markers with both myelomonocytic cells and T lymphocytes. Recently we described a new monoclonal antibody (MoAb), termed LAK1, which recognizes a 120 Kd surface molecule expressed on human large granular lymphocytes (LGL) and LAK precursors and effectors. LAK1 MoAb defines two different populations of positive cells amongst peripheral lymphocytes: the first subset (20%), represented by brightly stained cells, belongs to the non T-LGL population, whereas the second subset (30%) displays low fluorescence intensity and was partially composed of T lymphocytes. More interestingly, LAK1 is shared by some other cell types, such as monocytes and vascular endothelial cells. Immunohistochemical staining performed on muscle, endometrium and lymphoid or other non lymphoid tissues shows that LAK1 antigen is selectively expressed by the reticuloendothelial system.     

Confirmation of genital herpes simplex viral infection by an immunoperoxidase technique

Over the 12-month period from April 1984 to April 1985, 512,000 gynecologic (Papanicolaou) smears were examined in the Provincial Screening Program in British Columbia. During this time, 307 patients were found to have smears that contained cells consistent with, or suggestive of, a herpes simplex viral (HSV) infection. The Papanicolaou-stained smears from these 307 cases were subsequently restained, without prior destaining, using an immunoperoxidase technique specific for type 2 HSV (HSV-2) and cross reactive with HSV-1. Of the 205 smears containing cells considered to be consistent with a herpes infection, 187 were positive using the immunoperoxidase technique. Of the 102 smears showing reactive cell changes though unlikely to be causes by an HSV infection, only 5 were positive using the immunoperoxidase technique. The results show that the immunoperoxidase technique is a rapid and reliable method of confirming a suspected diagnosis of herpetic infection and that it is particularly useful in those patients in whom the Papanicolaou smear findings are equivocal.