Avidin-biotin Complex Technique Research Articles

Laser assisted microvascular anastomoses: A histological study

Laser assisted microvascular anastomosis (LAMA) was performed on rat femoral arteries anddd veins using a milliwatt CO2 laser. The histological appearance of the anastomoses was assessed immediately after the procedure and at regular intervals up to seven months. There was extensive thermal-induced transmural necrosis. The weld at the anastomosis was shown to comprise of fibrin, which later organized into fibrous tissue. Re-endothelialization occurred earlier in veins, and the endothelial nature of the surface cells was confirmed by factor VIII-related antigen positivity by the avidin-biotin peroxidase complex technique. Healing was complete by 9 weeks, but the vessel was not reconstituted to its normal appearance. At the site of the anastomosis 20% of the vessels had false aneurysms.

Expression of Vimentin and Epithelial Membrane Antigen in Human Malignant Lymphomas

Immunoreactivity with monoclonal antibodies against the intermediate filament protein, vimentin, and epithelial membrane antigen (EMA) was examined in 330 cases of lymphoma (317 non-Hodgkin's and 13 Hodgkin's lymphomas), 12 reactive lymph nodes and mononuclear cells of the peripheral blood using either indirect immunoperoxidase staining or the avidin-biotin immunoperoxidase complex technique. The cell origin of each tumor was established using a panel of monoclonal antibodies against lymphocyte differentiation antigens. There were 41 T-cell, 247 B-cell and 29 undetermined lymphomas, and 13 cases of Hodgkin's disease in the series. Vimentin was expressed in 24 T-cell lymphomas (58.5%) and 60 B-cell lymphomas (24.2%). This difference in frequency was statistically significant. Vimentin expression in follicular lymphomas was less frequent than in diffuse B-cell lymphomas. In diffuse lymphomas, small and medium cell types were more reactive with anti-vimentin than large cell types. Reed-Sternberg cells (R-S cells) in Hodgkin's disease were positive for vimentin in 11 cases (84.6%). The frequency of EMA reactivity in lymphomas was low, particularly in T-cell lymphomas. No positive cases were found among follicular lymphomas. In diffuse non-Hodgkin's lymphomas, EMA was expressed only in mixed and large cell types, but never in smaller ones. In conclusion, monoclonal antibodies against vimentin and EMA appear to be of limited usefulness for the diagnosis of non-Hodgkin's lymphomas, but anti-vimentin antibody may be used as an adjunct to the diagnosis of R-S cells in Hodgkin's disease.

Peptidergic innervation of the locus coeruleus cells in the human brain

The immunocytochemical localization of corticotropin-releasing factor (CRF), thyrotropin-releasing hormone (TRH), substance P (SP), neuropeptide Y (NPY), and neurotensin (NT) were studied in the human locus coeruleus (LC) using the avidin-biotin complex (ABC) technique. The brains were obtained from 3 adult male human subjects of 36–61 years old. All of these peptides studied were present in nerve fibers and varicosities throughout the entire nucleus, where they distributed unevenly. The highest density of immunoreactive neuronal networks was generally observed in the middle and to a lesser extent in the caudal part of the nucleus, while only scattered fibers were seen in the rostral part of the LC. Among the investigated neuronal peptides, the CRF-immunopositive network was the most dense, less dense immunostaining with TRH and NPY antisera while only few SP- and NT-immunoreactive fibers were present in the nucleus. Cell bodies immunostained for the above peptides were not seen in the LC. Per analogiam with rats, fibers immunostained in the LC may be of both intrinsic and extrinsic origins.

Topographic heterogeneity of amyloid B-protein epitopes in brains with various forms of neuronal ceroid lipofuscinoses suggesting defective processing of amyloid precursor protein

To verify our hypothesis of defective protease inhibitor domains that are encoded by abnormal processing of amyloid precursor protein (APP) in brains of patients with neuronal ceroid lipofuscinoses (NCL), immunohistochemical and cytochemical studies were performed with monoclonal antibodies (mAbs) directed against various domains of APP. For the studies, 22 autopsy brains were used: 12 with different forms of NCL, and 10 control brains. The staining procedure for the avidin-biotin complex (ABC) technique and the postembedding gold-labelled procedure for electron microscopy (EM) were employed. Of all mAbs used for the study, only mAbs generated against amyloid B-protein bound to neural tissue were affected with NCL. The strongest immunostaining of neurons and of some reactive glial cells was found in brains with the juvenile form of NCL. Only in the infantile form of the disease were some neurons overloaded with storage material weakly immunoreactive. In brains of patients with the adult form of NCL, immunoreactivity was found in affected neurons and in extracellularly deposited material of senile plaques. The results of EM study showed that the immunoreactivity was restricted to lysosomal cytosomes in neural tissue with any form of NCL selectively localized on the curvilinear and fingerprint proteinaceous component of ceroid lipofuscin. Studies performed on control aging brains and Alzheimer's disease (AD) brains confirmed previous observations of immunoreactivity being found diffusely in the protein component of some neurons containing lipopigment.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelial cells with vacuoles containing 54,000 dalton sialoglycoprotein in the mouse epididymal duct.

Epididymal ligation in the mouse induced unusual cells, called peculiar pale epithelial cells (hereafter, pale cells), specifically in the epithelium of the corpus epididymidis (ABE et al., 1982a, b). These pale cells had vacuoles with long microvilli on their inner surface. In this study, we found that the vacuoles of the pale cells occurred in normal mice and contained epididymal specific glycoprotein, sialoglycoprotein of 54,000 dalton (SGP54). This was elucidated by indirect immunofluorescence and avidin biotin complex techniques using monoclonal antibody T21 which specifically recognizes SGP54. These immunoreactive pale cells occurred in the distal caput and proximal corpus of the epididymidis. The relationship between the pale cell and SGP54 is discussed.

ImmunohistochemicaI and UItrastructuraI LocaIization of Epidermal Growth Factor Receptor in Human Liver and Hepatocellular Carcinoma Tissues

Expression of epidermal growth factor receptor (EGF-R) in human hepatocellular carcinoma (HCC), hepatoblastoma and non-cancerous liver tissues was investigated immunohistochemically in order to evaluate the possible role of EGF-R expression in neoplastic transformation of hepatocytes. Immunoreactive EGF-R molecules were identified on frozen sections by means of the avidin-biotin immunoperoxidase complex technique using a monoclonal antibody recognizing an epitope of the external domain of human EGF-R. Linear positive staining was present on the surface of carcinoma cells in one hepatoblastoma and in 9 of 11 HCCs. In addition, an enhanced level of surface EGF-R expression was observed on the tumor cells in 9 of 12 cases in comparison with that on hepatocytes in surrounding non-cancerous liver tissue, which in most cases showed chronic inflammation, hepatocyte injury or regeneration. No positive staining in the form of coalescent cytoplasmic granules was present in HCC or hepatoblastoma cells, nor in the cytoplasm of hepatocytes in normal or non-cancerous diseased liver tissue. Little or no reactivity was present on the surface membrane of hepatocytes in the normal liver tissues of 8 control cases. Furthermore, immunoelectron microscopy revealed the localization of this immunoreactive EGF-R molecule on the plasma membrane. Considering that the functional form of EGF-R could be localized on the plasma membrane, the enhanced expression of immunoreactive EGF-R on the tumor cell surface demonstrated here may suggest a possible role of EGF-R in the development or progression of human HCC as well as in hepatocyte regeneration.

A Comparison of a Simple Two Step Immunoalkaline Phosphatase Technique with the Peroxidase-Antiperoxidase (PAP) and Avidin Biotin Complex (ABC) Technique

Alkaline phosphatase enzyme is currently being used as a marker in immunopathology techniques. The system in this study uses the indirect alkaline phosphatase immunostaining technique known as the amplified substratelalkaline phosphatase (AS/AP™). This technique was compared to the widely used peroxidase-antiperoxidase (PAP) and avidinbiotin complex (ABC) techniques. The localization and demonstration of antigens were camed out on normal and neoplastic tissues fixed in formalin and routinely processed through paraffin wax.The results of this study indicate that the AS/AP™ technique is more sensitive (as much as ten times) than either the PAP or ABC technique. The increased sensitivity allows more economical use of expensive primary antibodies. The AS/AP™ technique produces a low background staining, intense specific immunostaining, and is simple to use with less hands on time. All of the reagents, including the chromogenic substrate, are provided in ready to use dropper bottles. (The J Histotechnol 12:279, 1989).

A Comparison of a Simple Two Step Immunoalkaline Phosphatase Technique with the Peroxidase-Antiperoxidase (PAP) and Avidin Biotin Complex (ABC) Technique

Alkaline phosphatase enzyme is currently being used as a marker in immunopathology techniques. The system in this study uses the indirect alkaline phosphatase immunostaining technique known as the amplified substratelalkaline phosphatase (AS/AP™). This technique was compared to the widely used peroxidase-antiperoxidase (PAP) and avidinbiotin complex (ABC) techniques. The localization and demonstration of antigens were camed out on normal and neoplastic tissues fixed in formalin and routinely processed through paraffin wax.The results of this study indicate that the AS/AP™ technique is more sensitive (as much as ten times) than either the PAP or ABC technique. The increased sensitivity allows more economical use of expensive primary antibodies. The AS/AP™ technique produces a low background staining, intense specific immunostaining, and is simple to use with less hands on time. All of the reagents, including the chromogenic substrate, are provided in ready to use dropper bottles. (The J Histotechnol 12:279, 1989).

Improved Sensitivity of Detection by Avidin—Biotin Complex (ABC) Immunocytochemistry in Epstein‐Barr Virus Serology

Serum antibodies against Epstein-Barr virus (EBV)-determined antigens have traditionally been titrated by the indirect immunofluorescence (IIF) technique. The avidin-biotin complex (ABC) immunocytochemical technique was used to determine the serum levels of IgA against EBV viral capsid antigen (IgA/VCA) and IgA against EBV early antigen (IgA/EA) in sera of 106 nasopharyngeal carcinoma (NPC) patients prior to treatment and 100 normal individuals. The sensitivity of the ABC technique is enhanced by an amplification of the antigen-antibody reaction, which involves the binding of the enzyme-linked ABC to the second biotinylated antibody. There was a good correlation (r = 0.9988) between ABC and IIF-determined IgA/VCA-positive titres, with the ABC technique being more sensitive than IIF in the detection of IgA/VCA in NPC sera: 94% (99/106) and 76% (80/106), respectively. The frequency of IgA/EA reactivity in NPC sera was also markedly increased by immunodetection with the ABC technique as compared with IIF technique: 63% (69/106) and 28% (30/106) respectively. Both the immunocytochemical techniques were equally specific in discriminating between elevated serum titres of IgA/VCA and IgA/EA in NPC sera from normal human sera.

Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods.

We developed an immunohistochemical method for visualization of vitamin D (VDR) and estrogen receptors (ER) in cryostat sections, using monoclonal antibodies (MAb) to the vitamin D receptor and estrogen receptor, respectively. This method is based on an avidin-biotin labeling technique (LAB). To establish a reliable and sensitive method which can be used easily as a routine diagnostic procedure, we systematically compared four different immunoenzymatic methods with respect to their efficiency in detecting vitamin D and estrogen receptors. Compared to the indirect bridged avidin-biotin (IBRAB), the peroxidase- anti-peroxidase (PAP), and the avidin-biotin complex (ABC) methods, the LAB method produced stronger staining intensities and had higher detection efficiency for both vitamin D and estrogen receptors. In addition, the LAB method had a higher spatial resolution compared to the ABC technique in detection of VDR in normal human skin biopsies. In the case of steroid receptors, i.e., nuclear antigens, immunohistochemistry must deal with a relatively low number of antigenic sites per cell, restricted accessibility of the antigens, and slight differences in antigen concentrations among cells. Under these particular conditions, the chemical properties of the conjugates used in the LAB method may explain why it is superior to the other methods. Consequently, the LAB method is recommended for visualization of ER and VDR.

Comparative quality assessment in immunocytochemistry: pilot study of CD15 staining in paraffin wax embedded tissue in Hodgkin's disease.

The feasibility of comparative quality assessment studies in immunocytochemistry was examined. The reactions of three CD15 antibodies--anti-Leu M1, DM1, and Tü9--were examined in paraffin wax sections in Hodgkin's disease under a variety of different fixation and pre-treatment conditions, using four immunochemical detection techniques. All three antibodies stained Reed-Sternberg cells, but DM1 could be used at slightly higher dilutions to achieve comparable results. Tissue fixed in formol sublimate showed the most intense staining reactions, and formol saline and neutral buffered formalin gave relatively poor results. Although neuraminidase pre-treatment improved staining, its routine use is probably contraindicated by its high cost. Trypsinisation has some value for sections of tissue fixed in formol saline and neutral buffered formalin. The avidin-biotin complex technique produced the best results, but indirect immunoperoxidase produced acceptable results, is technically easier to perform, and is less expensive. It is concluded that information regarding variations in techniques and commercially available reagents, which may be of use in routine diagnostic histopathology, can be obtained by comparative quality assessment studies of this type.

Immunohistochemical expression of neuron-specific enolase and Leu 7 in Ewing's sarcoma of bone.

Neural features have been documented in a series of small round cell tumors of bone, previously classified as Ewing's sarcoma of bone (ESB), using light microscopic, ultrastructural, immunohistochemical, and in vitro techniques. To date, correlation of the presence of these features in ESB with clinical outcome has not been performed. The authors investigated the clinical relevance of positivity to antibodies against neuron-specific enolase (NSE) and Leu 7 (HNK 1) using the avidin-biotin complex technique in 40 cases of ESB seen at St. Jude (Memphis, TN) during the period 1968 to 1986. Twenty-three cases (58%) were positive for NSE and/or Leu 7. The median disease-free survival of patients with localized tumors and NSE and/or Leu 7 positivity was 3.8 years compared to a median disease-free survival of 1.6 years for those without these markers and localized disease. Using the log-rank test, a statistically significant difference was shown in these two groups of patients (P = 0.049). The authors conclude that in this relatively small series ESB commonly expresses NSE and/or Leu 7 and that the presence of these markers is of no prognostic significance overall, but that in patients with localized ESB the presence of NSE and/or Leu 7 by immunostaining may be a favorable prognostic indicator.

Immunofluorescent analysis of estrogen induction of progesterone receptor in the rhesus uterus.

Estrogen (E) has been shown to induce an increase in progesterone (P) receptor (PR) concentration in uterine tissue of both rodents and primates. Because of the presence of different cell types within the uterus, we were interested in determining whether estrogen-induced PR were cell type specific in the nonhuman primate uterus (rhesus monkey). Immunofluorescent analyses of E receptor (ER) and PR were performed on fresh frozen cryostat sections (6 microns) of uterine tissue from ovariectomized (3 months) and estradiol (E2)-treated (peak level of E2 during an artificial menstrual cycle) rhesus monkeys. Antibodies to ER and PR were obtained from Abbott Laboratories (H222) and Transbio (MPR1). The avidin-biotin complex technique was used with streptavidin-conjugated Texas red for fluorescent detection. Ovariectomized monkeys showed positive fluorescence for ER in luminal and glandular epithelia, stromal cells, and smooth muscle cells of the myometrium. In contrast, positive fluorescence for PR was observed primarily in glandular epithelia, with little or no fluorescent detection in luminal epithelium, stromal cells, or myometrial smooth muscle cells. After E2 treatment strong positive fluorescence for PR was observed in luminal and glandular epithelia, stromal cells, and myometrial smooth muscle cells. Strong positive fluorescence for ER was also observed in the same cell types. Fluorescent detection of ER and PR was restricted to the nuclei of these cell types. These studies show that ER are present constitutively in all cell types of the E-withdrawn (ovariectomized) nonhuman primate uterus, whereas PR are primarily restricted to glandular epithelia. E2 treatment, which simulated the follicular phase and E2 surge, resulted in the appearance of immunofluorescent PR in luminal epithelia, stromal cells, and myometrial smooth muscle cells. These studies serve to define the cellular pattern of E2-induced PR in the primate uterus.

ABO blood grouping of hairs using an avidin-biotin-peroxidase complex technique.

Fifty-nine hair specimens obtained from human autopsies and volunteers were used for the determination of ABO blood group substances using the ABC (Avidin-Biotin Complex) technique. Positive staining for A, B and H blood group substances was detected only in the medulla of the hairs. Blood group antigens could not be detected in seven hair specimens because they possessed no medulla. Forty-seven specimens obtained from fresh cadavers and volunteers gave the correct results corresponding to the blood group of the donor, but some specimens from individuals of blood group A2, Le(a + b-) showed weak reaction with anti-A and strong reaction with anti-H. The staining intensity with anti-B and -H in some individuals of blood group AB was stronger than with anti-A serum. Five hair specimens obtained from decomposed bodies were also examined. The blood group antigens could be specifically detected in hairs obtained from two exhumed and one putrid body, but no positive reactions were obtained from two cases of drowning where the bodies had been in the sea for about 6 months. In a blind trial, hair specimens from 28 individuals were also examined. Twenty-two specimens which possessed a medulla gave the correct result. Six specimens gave no result because they possessed no medulla.

A Rapid Immunohistologic Method for Determining the Limit of Cancer Invasion at the Surgical Margin of Scirrhous Carcinoma of the Stomach During Surgery

The authors used the monoclonal antibody (MAb) S202 as an adjunct for detection of scirrhous gastric carcinoma cells in cytomorphologic preparations of the resected stomach. Twenty-six samples of scirrhous gastric cancer were stained by the avidin-biotin complex technique on fixed frozen sections. MAb S202 was strongly reactive on all sections. In 12 cases, samples obtained from surgical margins were examined by a rapid immunostaining method. In one case, conventional cytologic findings were equivocal, whereas by the rapid immunostaining method single cells were stained darkly, indicating malignancy. Thus, immunohistochemical analysis using MAb S202 should be carried out to determine the limit of invasive carcinoma at the time of surgery.

Subacute toxicity of (-)15-deoxyspergualin in BALB/c mice. II. Histopathological study.

Following the preceding report on hematological aspects in subacute toxicity of (-)15-deoxyspergualin (DSP), the present report dealt with the histopathological changes produced by DSP, 0.5-5.0 mg/kg, given to BALB/c mice for three months. At sacrifice, various internal organs such as heart, lungs, liver, spleen and kidneys, were taken, and their wet weights immediately measured. HE- or PAS-stained sections were histopathologically studied under light microscope. Additionally, frozen sections of the spleen were prepared to evaluate the effect of DSP on the lymphocyte surface markers like thy 1 and B220 by avidin-biotin complex (ABC) technique. Although the mean weights of lungs, liver, spleen and kidneys in the 0.5 mg- and 2.5 mg-DSP groups were not significantly different from those in the control animals, the weights of the heart, lungs, liver and spleen in the 5 mg-DSP group were significantly lower. Histopathological studies by light microscopy revealed no abnormalities in the heart, lungs, liver or kidneys taken from the mice given 0.5-5.0 mg/kg DSP. In contrast, significant changes were observed in the spleen and bone marrow of the 5.0 mg group of mice. Likewise, in the intestine of the 0.5-5.0 mg groups dose-dependent lesions, such as degeneration or disappearance of the mucosal epithelium, infiltration by inflammatory cells, and pseudo-membrane formation, was observed. By ABC technique, preferential decrease of B cells was seen in the splenic corpuscles of the DSP-treated mice. Histopathological changes due to DSP predominantly seen in the lymphoid and/or hematopoietic organs may be directly related to the immunosuppressive potency inherent to this drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical detection of luteinizing hormone in epididymis of mature mouse

Luteinizing hormone (LH) binds to the Leydig cells of several mammalian species where it stimulates steroidogenesis, protein synthesis and protein phosphorylation. In the present study, standard immunoperoxidase (PAP) and avidin-biotin complex (ABC) techniques were used to detect the binding of endogenous and exogenous LH to the epididymis of the mature mouse. Throughout the epididymal duct, a positive reaction for peroxidase, indicating LH binding, occurred in the Golgi area of principal cells. In segment 1, positive reactions were also visualized in the perinuclear area and in the region located between the Golgi area and the apical surface of the principal cells (supra-Golgi area). In the corpus and cauda epididymidis, scattered entire principal cells were also positive. Throughout the epididymal duct, the reactions indicating the binding of exogenous LH were more intense than those of endogenous LH. The significance of LH binding to the epididymis is uncertain but LH may perform the same functions in epididymal principal cells as it does in Leydig cells.

Intermediate Filament Staining in the Cytologic and Histologic Diagnosis of Canine Skin and Soft Tissue Tumors

Formalin-fixed histologic and acetone-fixed cytologic preparations from 87 surgically removed subcutaneous and soft tissue canine tumors were examined for immunoreactivity to cytokeratin, desmin, and vimentin. The avidin-biotin complex (ABC) technique demonstrated immunoreactivity in both preparations, but the intensity and specificity of the reactions were dependent on the primary antibody. Polyclonal antibodies to cytokeratin were more consistent in immunoreactivity than were polyclonal desmin or vimentin antibodies. The monoclonal antibody proved more satisfactory for demonstrating vimentin than the polyclonal antibody. Greater dilutions of primary antibodies may be used on cytologic preparations than on histologic sections. Evaluation of cytologic preparations may be inconclusive due to background staining, scant cellularity, or poor cytoplasmic preservation.

Localization of corticotropin-releasing factor, somatostatin, and vasoactive intestinal polypeptide in the parabrachial nuclei of the human brain.

The immunocytochemical localizations of corticotropin-releasing factor (CRF), somatostatin (SRIF), and vasoactive intestinal polypeptide (VIP) were studied in the human parabrachial nuclei (PBN) using the avidin-biotin complex (ABC) technique. The brains were obtained from seven adult male human subjects of 38-74 years. In three cases, the brains were fixed within 2 hr, in four cases within 5 hr, postmortem. All of these peptides were detected in fibers through the orocaudal extent of the lateral PBN, whereas the medial nucleus contained only CRF immunoreactive fibers. Immunoreactive fibers were distributed unevenly within the lateral nucleus with the highest density in the dorsal and much fewer in the ventral part of the lateral subdivision. The highest to lowest density of immunostained processes were detected using CRF, SRIF, and VIP antisera, respectively. Since NPB is known as an important relay nucleus for the central autonomic pathway, the presence of the above noted neuropeptides in nerve fibers in this area may suggest a neurotransmitter or neuromodulatory role of CRF, somatostatin, and VIP in certain autonomic nervous mechanism of the human brain.

Immunohistochemical staining for terminal deoxynucleotidyl transferase (TDT). An enhanced method in routinely processed formalin-fixed tissue sections.

This report describes an improved technique for sensitive and specific localization of terminal deoxynucleotidyl transferase (TdT) in routinely processed paraffin-embedded, formalin-fixed tissue sections using DNAse pretreatment and the avidin-biotin complex (ABC) technique. This method is useful in identifying lymphoblastic lymphomas (14/15 cases positive), with all other B- and T-cell lymphomas tested negative for the reaction. Used in conjunction with monoclonal antibodies immunoreactive for T- and B-cells in paraffin sections this technique should prove helpful in immunophenotyping malignant lymphomas where fresh tissue is unavailable for study.