scholarly journals Comparison of paired immunofluorescence and paired immunoenzyme staining methods based on primary antisera from the same species.

1982 ◽  
Vol 30 (6) ◽  
pp. 518-524 ◽  
Author(s):  
K Valnes ◽  
P Brandtzaeg
Keyword(s):  
Model Systems ◽  
Detection Sensitivity ◽  
Direct Immunofluorescence ◽  
Tissue Sections ◽  
Staining Methods ◽  
Pap Method ◽  
Blue Reaction ◽  
Antigen Antibody ◽  
Unlabeled Antibody ◽  
Sufficient Strength

Evaluation of sequential paired staining with the unlabeled antibody peroxidase-antiperoxidase (PAP) method showed that the brown reaction product of diaminobenzidine (DAB) concealed both enzyme and antigen-antibody sites in the reagent sequence. The blue reaction product of the alternative substrate, 4-chloro-1-naphthol (CN), exerted no such blocking effect. Hence, to avoid interactions between the two PAP sequences, DAB had to be used for the first and CN for the second antigen. Complete blocking required that the DAB color reaction be of sufficient strength. When two antigens were present in the same cell, the DAB deposits inhibited staining of the second antigen unless the brown color was decreased by progressive dilution of the initial primary antibody. A mixture of brown and blue could thus reflect either concomitant staining of the two antigens or unwanted interactions between the two PAP sequences. Double staining of individual cells was, therefore, equivocal and conclusions had to be based on comparative single staining results in adjacent tissue sections. Tests carried out in several model systems showed that paired direct immunofluorescence with fluorochrome conjugates of contrasting colors (green and red) was much less time-consuming, more reliable, and of higher detection sensitivity for analyses of unbalanced mixtures of two antigens in the same cell.

scholarly journals Unlabeled antibody peroxidase-antiperoxidase method combined with direct immunofluorescence.

1981 ◽  
Vol 29 (6) ◽  
pp. 703-711 ◽  
Author(s):  
K Valnes ◽  
P Brandtzaeg
Keyword(s):  
Epithelial Transport ◽  
Secretory Component ◽  
Secretory Iga ◽  
Direct Immunofluorescence ◽  
Myeloma Cells ◽  
Quenching Effect ◽  
Blocking Effects ◽  
Pap Staining ◽  
Pap Method ◽  
Unlabeled Antibody

Paired staining with the unlabeled antibody peroxidase-antiperoxidase (PAP) method and direct immunofluorescence (DIF) differentiated distinctly between gastrin- and somatostatin-producing cells in the human gastric antrum. Similar paired staining of complexed lambda and alpha chains in immunoglobulin (Ig)A myeloma cells, of kappa and free J chains in IgG myeloma cells, and of secretory IgA and its epithelial transport protein, the free secretory component (SC), in colonic crypt cells, demonstrated that PAP staining inhibits subsequent DIF staining of an antigenic determinant present on the same molecule as the antigen revealed by the brown color of diaminobenzidine (DAB) or present or an unassociated molecule in the same cell. A quenching effect of the DAB reaction product was noted for both fluorescein (green) and rhodamine (red) emissions. In addition, a blocking effect of the DAB deposits has been demonstrated and is assumed to be the principal methodological basis for the paired PAP-DIF staining approach omitting intermediate antibody elution, as well as for the more time-consuming sequential PAP staining with DAB substrate for the first and 4-chloro-1-naphthol (CN) for the second antigen. The quenching and blocking effects limit in practice the paired PAP-DIF method to the localization of antigens present in separate cells.

scholarly journals Protein A, avidin, and biotin in immunohistochemistry.

1981 ◽  
Vol 29 (11) ◽  
pp. 1349-1353 ◽  
Author(s):  
S M Hsu ◽  
L Raine
Keyword(s):  
Plasma Cells ◽  
Protein A ◽  
Intense Staining ◽  
Background Staining ◽  
Main Disadvantage ◽  
Pap Method ◽  
Abc Method ◽  
Antigen Antibody ◽  
Unlabeled Antibody ◽  
Ig G

Conjugated and unlabeled peroxidase antibody methods have proven to be quite satisfactory in localizing sites of antigen-antibody reaction. The use of avidin-biotin-peroxidase complex (ABC), as well as protein A, can contribute significantly to the field of immunohistochemistry. The sensitivity and specificity of several immunohistochemical methods is compared. In general, the ABC method produced the most intense staining and the least background staining of any method tested. The unlabeled antibody (peroxidase-antiperoxidase: PAP) method also yielded satisfactory results, but it was less intense than the ABC method. In comparison to the PAP method, the indirect conjugated method presented slightly inferior staining intensities and significantly higher background staining. Protein A techniques produced a range of staining sensitivities similar to or inferior to the PAP technique. The main disadvantage in using protein A is that it reacts with intrinsic immunoglobulin (Ig) G, thus producing an intense background. Therefore, its use is not recommended on tissues that have either abundant immunoglobulins in their interstitium or numerous IgG-containing plasma cells.

scholarly journals The unlabeled antibody method: comparison of peroxidase-antiperoxidase with avidin-biotin complex by a new method of quantification.

1986 ◽  
Vol 34 (5) ◽  
pp. 599-605 ◽  
Author(s):  
L A Sternberger ◽  
N H Sternberger
Keyword(s):  
Method Comparison ◽  
Staining Intensity ◽  
The Other ◽  
Tissue Sections ◽  
Low Concentrations ◽  
Pap Method ◽  
Abc Method ◽  
Straight Lines ◽  
Unlabeled Antibody ◽  
Avidin Biotin Complex

Upon plotting of areas against optical densities in immunocytochemically stained tissue sections, hyperbolic curves were obtained which could be reduced to two straight lines, one representing variations in stained structures, and the other variations in background. The slopes of the stained structure lines reflected staining intensity independently of total area of stained structure in a section. The ratio of slopes of the stained structure and background lines reflected immunocytochemical sensitivity. A comparison of the peroxidase-antiperoxidase (PAP) method with the avidin-biotin complex (ABC) method showed that at usual antibody dilutions the PAP method was much more sensitive than the ABC method, while at impractically high antibody dilutions it was moderately more sensitive. Once sufficient dilutions of antibodies were reached, staining intensities dropped sharply with the PAP method. On the other hand, the dilution curves were flat with the ABC method. The ABC method consequently appeared unsuitable for estimating variations in concentration of antigen or for distinguishing high or low concentrations of antigen. The ABC method provided a stain for myelin even in the absence of any antibodies.

scholarly journals Immunocytochemical localization of Mullerian inhibiting substance in the rough endoplasmic reticulum and Golgi apparatus in Sertoli cells of the neonatal calf testis using a monoclonal antibody.

1984 ◽  
Vol 32 (6) ◽  
pp. 649-654 ◽  
Author(s):  
M Hayashi ◽  
H Shima ◽  
K Hayashi ◽  
R L Trelstad ◽  
P K Donahoe
Keyword(s):  
Monoclonal Antibody ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rough Endoplasmic Reticulum ◽  
Sertoli Cells ◽  
Neonatal Calf ◽  
Müllerian Inhibiting Substance ◽  
Pap Method ◽  
Unlabeled Antibody ◽  
Mullerian Inhibiting Substance

Mullerian Inhibiting Substance (MIS) has been localized in the Sertoli cells of the neonatal calf testis using preembedding immunoperoxidase techniques and a monoclonal antibody which almost completely blocks the biological activity of MIS. Both the peroxidase-labeled antibody method using a peroxidase-conjugated F(ab')2 fragment of IgG as a second antibody and the unlabeled antibody peroxidase-antiperoxidase (PAP) method using Fab fragments of the PAP complex were employed. With both methods, MIS was demonstrated within the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus. In the Golgi, MIS was concentrated in the transmost cisternae especially at their peripheral expansions. This study indicates that MIS is synthesized in the RER and transported to the Golgi apparatus, presumably for glycosidation, before secretion from Golgi derived vacuoles.

scholarly journals Localization of the loosely bound nuclear proteins of rat brain: an immunocytochemical ultrastructural study.

1980 ◽  
Vol 28 (6) ◽  
pp. 552-556 ◽  
Author(s):  
D Cocchia ◽  
F Michetti
Keyword(s):  
Rat Brain ◽  
Active Regions ◽  
Antigenic Determinants ◽  
Brain Nuclei ◽  
Liver Nuclei ◽  
Pap Method ◽  
The One ◽  
Unlabeled Antibody ◽  
The Brain ◽  
Isolated Liver

Antibodies against the loosely bound subnuclear protein fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the ultrastructural distribution of the antigenic determinants in rat cerebellum was studied using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. A localization restricted to the nucleus was observed. Both neuronal and neuroglial nuclei exhibited antigens, whereas nuclei of pericytes and endothelial cells did not. The immunoreaction product was homogeneously distributed in dispersed chromatin and was absent from condensed chromatin, suggesting that the antigens were confined to the active regions of the genoma. The outer nuclear membrane and the nucleolus appeared to be free of the antigens, while a perinucleolar ring of immunoreaction was detectable. Liver preparations showed a nuclear reaction markedly weaker than the one for brain nuclei. Adsorption of the serum with isolated liver nuclei nullified the reactivity in liver tissue, whereas a sharp reaction was still observed in the cerebellum, indicating the subcellular reaction under examination to contain antigens specifically concentrated in the nervous system or unique to the brain.

scholarly journals Ultrastructural immunohistochemical localization of anti-invasion factor (AIF) in bovine cartilage matrix.

1982 ◽  
Vol 30 (6) ◽  
pp. 524-531 ◽  
Author(s):  
F H Wezeman ◽  
G V Childs
Keyword(s):  
Staining Technique ◽  
Ultrastructural Localization ◽  
Immunohistochemical Localization ◽  
Cartilage Matrix ◽  
Fixed Tissue ◽  
Antibody Complex ◽  
Protein Components ◽  
Antigen Antibody ◽  
Unlabeled Antibody ◽  
Bovine Cartilage

Rabbit antibodies prepared against bovine cartilage anti-invasion factor (AIF) were tested for their affinity toward antigenic sites in glutaraldehyde-fixed bovine hyaline cartilage matrix. Ultrastructural localization of the antigen-antibody complex was accomplished by the unlabeled antibody peroxidase-antiperoxidase staining technique. Unextracted and salt-extracted (1 M NaCl or 3 M GuHCl) cartilage slices were incubated with anti-AIF antibodies at a working dilution of 1:20,000. Staining occurred in unextracted matrix distributed throughout the tissue, but with regional variation in the lacunar matrix. Significantly less stain was noted in extracted tissues. The results suggest that at least certain protein components in AIF are morphologically associated with matrix complexes in aldehyde-fixed tissue.

scholarly journals Immunocytochemical localization of the posterior lobe hormones and of their carrier proteins.

1980 ◽  
Vol 28 (5) ◽  
pp. 469-471 ◽  
Author(s):  
F Vandesande ◽  
K Dierickx ◽  
N Goossens
Keyword(s):  
Staining Method ◽  
Posterior Lobe ◽  
Serial Sections ◽  
Double Staining ◽  
Carrier Proteins ◽  
Tissue Sections ◽  
Tissue Antigens ◽  
Staining Methods ◽  
Double Staining Method ◽  
The One

Serial sections of vertebrate hypothalami were stained with the immunocytochemical peroxidase-antiperoxidase method. In addition to the single staining method, our double staining method was used, which enabled us to visualize two tissue antigens in single tissue sections. In both staining methods, differentially adsorbed antineurohypophysial hormone sera, anti-somatostatin serum and anti-bovine neurophysin sera were used. The results confirm the one hormone, one neuron hypothesis.

scholarly journals Lactate dehydrogenase isozymes in developing rat oral mucosa. A comparative study of LDH biochemistry and histochemistry.

1984 ◽  
Vol 32 (9) ◽  
pp. 958-964 ◽  
Author(s):  
S Sjögren
Keyword(s):  
Basal Layer ◽  
Point Of View ◽  
Oral Epithelium ◽  
Lactate Dehydrogenase Isozymes ◽  
Tissue Sections ◽  
Ldh Activity ◽  
Staining Methods ◽  
Cyanide Inhibition ◽  
Ldh Assay ◽  
Developing Rat

Histochemical lactic acid dehydrogenase (LDH) staining methods seem unable to demonstrate the total LDH activity in tissue sections. An analysis was made of LDH tissue staining methods applied on LDH zymograms. The menadione-mediated LDH staining of tissue sections can not possibly reflect true LDH activity. The addition of cyanide also slightly inhibited LDH activity. The cyanide inhibition was confirmed via LDH assay and found to be competitive in character. It is concluded that cyanide and menadione should be replaced by agents suitable from both a histochemical and a biochemical point of view. Based on the findings of this study the presence of LDH in oral epithelium was analyzed. Evidently LDH of the oral epithelium is basically anaerobic in character and located primarily in spinosum/granulosum layers and only sparsely in the basal layer.

Combined application of monoclonal antibody to human la antigens and avidinblotin-peroxidase staining method in immunoelectron microscopy

1984 ◽  
Vol 42 ◽  
pp. 258-259
Author(s):  
K.C. Feng-Chen ◽  
B.F. Chen ◽  
A.K. Ng
Keyword(s):  
Monoclonal Antibody ◽  
Immunoelectron Microscopy ◽  
Staining Method ◽  
Ultrastructural Level ◽  
Combined Application ◽  
Antibody Method ◽  
Tissue Antigens ◽  
Pap Method ◽  
Abc Method ◽  
Unlabeled Antibody

Immunological detection of cellular and tissue antigens have been based on the widely used original enzyme labeled antibody method or the unlabeled antibody, peroxidase-anti-peroxidase (PAP) method. Recently, an improved immunoenzymatic technique for light microscopy using the avidin-biotinperoxidase complex (ABC) system was developed for cell and tissue staining, and found to be superior to the PAP technique in terms of sensitivity and specificity. The application of the ABC method in immunoelectron microscopy (IEM), however, has not been extensively evaluated. This study demonstrates that the ABC method is suitable for localizing specific cellular antigen at the ultrastructural level with monoclonal antibody (MAb), the production of which by somatic cell hybridization is now a well established procedure.

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