scholarly journals Immunohistochemical staining on hydroxyethyl-methacrylate-embedded tissues.

1983 ◽  
Vol 31 (8) ◽  
pp. 1000-1004 ◽  
Author(s):  
S Casanova ◽  
U Donini ◽  
N Zini ◽  
R Morelli ◽  
P Zucchelli
Keyword(s):  
Extracellular Protein ◽  
Renal Amyloidosis ◽  
Hydroxyethyl Methacrylate ◽  
Frozen Sections ◽  
Protein Antigens ◽  
Biopsy Tissue ◽  
Background Staining ◽  
Ig G ◽  
Lower Background ◽  
Trace Reactions

Hydroxyethyl-methacrylate (GMA) embedding has recently been proposed for light microscopy studies. In the present investigation extracellular protein antigens were localized on GMA-embedded renal biopsy tissue. Conventionally frozen sections were compared with GMA sections from 55 renal specimens for the detection of extracellular protein antigens. Sections were directly stained with fluorescein- or peroxidase-conjugated antisera against immunoglobulin (Ig) G, IgA, IgM, C3, C1q, and fibrinogen. Results obtained using these two methods showed a 74-89% agreement, depending on the antigen under study. Some discrepancy between GMA and frozen sections was observed in three cases of renal amyloidosis and those cases presenting focal or trace reactions; the differences did not, however, influence the diagnosis. Prerequisites for antigen recovery on GMA sections were a) choice of fixative; b) abrupt dehydration of specimens; and c) treatment of sections with nonspecific protease. The improved localization and the lower background staining obtained led to easy and immediate detection of antigens on GMA sections despite the reduced antigenicity due to the embedding process.

The Application of Ultracryotomy to Immunocytochemistry

1973 ◽  
Vol 31 ◽  
pp. 704-709
Author(s):  
R. G. Painter ◽  
K. T. Tokuyasu ◽  
S. J. Singer
Keyword(s):  
Frozen Sections ◽  
Protein Antigens ◽  
Carbon Coated ◽  
Antibody Conjugates ◽  
Ultrathin Frozen Sections

A technique for localizing intracellular antigens with immunoferritin conjugates directly on ultrathin frozen sections of glutaraldehyde-fixed tissues has been developed. This method overcomes some of the limitations of previously described procedures, since it avoids drastic fixation, dehydration and embedding procedures which could denature many protein antigens.Briefly cells or tissues were fixed with glutaraldehyde (0.5 to 2% for 1 hr), and ultrathin frozen sections were cut and mounted on grids covered with carbon-coated Formvar film by the procedure described previously. Such sections were stained with ferritin-antibody conjugates by methods described elsewhere.

scholarly journals IMMUNOGENIC PROPERTIES OF CELLULAR AND EXTRACELLULAR PROTEIN-CONTAINING ANTIGENS OF STAPHYLOCOCCUS AUREUS

2019 ◽  
Vol 1 (1) ◽  
pp. 29-36
Author(s):  
I. M. Gruber ◽  
N. B. Egorova ◽  
E. A. Astashkina ◽  
N. K. Akhmatova ◽  
E. A. Kurbatova ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Innate Immunity ◽  
Cell Wall ◽  
Virulent Strain ◽  
Surface Antigens ◽  
Extracellular Protein ◽  
Mononuclear Leukocytes ◽  
Protective Activity ◽  
Protein Antigens ◽  
Experimental Drugs

Aim. Comparative study of immunobiological properties of cell wall surface antigens and extracellular protein-containing antigens of Staphylococcus aureus. Materials and methods. Preparations: surface antigens of the cell wall (peptidoglycan, teichoic acids, protein antigens) of the strains of S. aureus containing in the staphylococcal vaccine «Staphylovac» (SV) and extracellular protein-containing antigens of S. aureus (EPCA). The parameters of innate immunity were evaluated by the effect of preparations on the immunophenotype of mononuclear leukocytes (ML) of the spleen of mice, expression of Toll-like receptors (using flow cytometry), phagocytic activity of macrophages of peritoneal exudate of mice after the introduction of SV and EPCA; the protective activity of preparations was studied in experiments of active protection of BALB/c mice. Results. To isolate EPCA, we used the virulent strain of S. aureus №6, and for obtaining surface antigens of the cell wall, 4 strains were selected, the most immunogenic of which was the low virulent strain of S. aureus №1991. Both preparations increased the number of TLR2 and MHC II expressing cells; CV administration caused an increase in the number of cells with CD25 marker, reflecting the early activation of immunocompetent cells, and EPCA immunization — led to a shorter expression of this marker. On the other hand, an increased number of CD19 positive cells were detected for a longer period of time during EPCA immunization. The longest activation of phagocytosis under the action of SV was established. High protective activity of bo-th types of the studied preparations was noted. Conclusion. The antigens of the cell wall and extracellular protein-containing experimental drugs possessed comparable immunological  properties, however, the surface antigens to a greater extent activate innate immunity, and extracellular — adaptive immunity.

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.

Immunocytochemical localization of nerve growth factor: effects of fixation.

1984 ◽  
Vol 32 (1) ◽  
pp. 30-36 ◽  
Author(s):  
D J Hazen-Martin ◽  
J A Simson
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Microscopic Level ◽  
Frozen Sections ◽  
Light Microscopic Level ◽  
Paraffin Sections ◽  
Nerve Growth ◽  
Background Staining ◽  
Diffuse Background ◽  
Chloroform Methanol

The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.

scholarly journals Mixed glycosidase pretreatment reduces nonspecific binding of antibodies to frozen tissue sections.

1985 ◽  
Vol 33 (7) ◽  
pp. 695-698 ◽  
Author(s):  
L P Andrews ◽  
R K Clark ◽  
I Damjanov
Keyword(s):  
Monoclonal Antibody ◽  
Renal Tissue ◽  
Frozen Sections ◽  
Tissue Sections ◽  
Mouse Tissues ◽  
Background Staining ◽  
Frozen Tissue ◽  
Nonspecific Staining ◽  
Immunohistochemical Studies ◽  
Monoclonal Antibody Binding

Indirect immunohistochemical studies of frozen mouse tissues with mouse monoclonal antibodies yield, in general, suboptimal results primarily because of indiscriminate binding of secondary antibody to all mouse immunoglobulins, i.e., to the monoclonal reagent and to endogenous immunoglobulin nonspecifically trapped in the tissue. To reduce this nonspecific staining, frozen sections of mouse kidney were treated enzymatically. Optimal results were obtained following a 2 hr treatment with 20 mg/ml of mixed glycosidases (MG). This treatment reduced the nonspecific background staining of the interstitial spaces and blood vessels, but did not affect the reactivity of structurally bound immunoglobulin G (IgG) in the glomeruli or alter the reactivity of mouse renal tissue to the monoclonal antibody that recognizes an oligosaccharide antigenic determinant (SSEA-1). Eluates from enzyme-treated frozen tissue sections contained normally immunoreactive IgG in the form of dimers. These data indicate that MG treatment of frozen sections could be safely used to reduce the content of nonstructurally bound immunoglobulins in frozen tissues and thus improve the visualization of specific monoclonal antibody binding.

scholarly journals Antigen-Specific Urinary Immunoglobulin in Reservoir Hosts of Leptospirosis

2021 ◽  
Vol 8 (9) ◽  
pp. 178
Author(s):  
Jarlath E. Nally ◽  
Richard L. Hornsby ◽  
David P. Alt
Keyword(s):  
Urinary Excretion ◽  
Animal Species ◽  
Renal Excretion ◽  
Clinical Manifestations ◽  
Domestic Animals ◽  
Protein Antigens ◽  
Cellular Immune ◽  
Reservoir Hosts ◽  
Ig G ◽  
Biological Equilibrium

Domestic and wildlife animal species act as reservoir hosts of leptospirosis, a global zoonotic disease affecting more than 1 million people annually and causing significant morbidity and mortality in domestic animals. In contrast to incidental hosts which present with an array of clinical manifestations, reservoir hosts are typically asymptomatic and can shed leptospires from chronically infected kidneys via urine for extended periods of time. Renal excretion of leptospires occurs despite evidence of a humoral and cellular immune response and is reflective of the unique biological equilibrium that exists between certain animal species and specific serovars of Leptospira. Here, we demonstrate that urinary excretion of leptospires is accompanied by the presence of antigen-specific urinary immunoglobulin. In rats experimentally infected with L. interrogans serovar Copenhageni using the intraperitoneal or conjunctival route of inoculation, urinary immunoglobulin (Ig) G specific for protein antigens was detectable within 1 week. Rat urinary IgG was not bound to urinary-derived leptospires. In cattle that were naturally exposed to, and infected with, L. borgpetersenii serovar Hardjo, urinary IgA specific for protein antigens was detected. Collectively, these results demonstrate that urinary excretion of immunoglobulin specific for leptospires is a hallmark of reservoir hosts of infection.

scholarly journals Improved procedures for immunoferritin labeling of ultrathin frozen sections.

1976 ◽  
Vol 71 (3) ◽  
pp. 894-906 ◽  
Author(s):  
K T Tokuyasu ◽  
S J Singer
Keyword(s):  
Cross Linking ◽  
Frozen Sections ◽  
Glutaraldehyde Fixation ◽  
Protein Antigens ◽  
Specific Staining ◽  
Ultrathin Sections ◽  
Ultrathin Frozen Sections ◽  
Structural Preservation

In employing fixed frozen ultrathin sections as substrates for immunoferritin labeling of intracellular antigens, we have found that conventional glutaraldehyde fixation sometimes permits very little specific staining of the sections, either because it inactivates certain protein antigens, or because it renders them inaccessible to the antibody stains. We have developed several fixation procedures that are chemically milder and allow a uniform but less extensive cross-linking of the specimen. With these procedures and precautions in the handling of the more fragile frozen sections, excellent structural preservation and specific immunoferritin labeling has been achieved with several systems.

scholarly journals Paraffin Immunofluorescence Increases Light-Chain Detection in Extra-Renal Light Chain Amyloidosis and Other Light-Chain–Associated Diseases

2020 ◽  
Author(s):  
Jean-Baptiste Gibier ◽  
Romain Perbet ◽  
Benjamin Lopez ◽  
Magali Colombat ◽  
Romain Dubois ◽  
...  
Keyword(s):  
Light Chain ◽  
Retrospective Cohort ◽  
Staining Intensity ◽  
Renal Diseases ◽  
Direct Immunofluorescence ◽  
Renal Amyloidosis ◽  
Frozen Sections ◽  
Light Chain Amyloidosis ◽  
Associated Diseases ◽  
Formalin Fixed Paraffin Embedded

Context.— Distinguishing the different types of amyloid is clinically important, because treatments and outcomes are different. Mass spectrometry is the new gold standard for amyloid typing, but it is costly and not widely available. Therefore, immunolabeling remains the first step in identifying the most common types of amyloidosis. In amyloid subtyping, direct immunofluorescence works well when applied to frozen sections, but immunohistochemistry on formalin-fixed, paraffin-embedded material often yields poor results, particularly for light chain amyloidosis. Recently, paraffin immunofluorescence has been described as a valuable salvage technique in renal pathology when frozen sections are not available but it has not been evaluated for extra-renal diseases. Objectives.— To evaluate the use of paraffin immunofluorescence for light-chain detection in extra-renal amyloidosis and other light-chain–associated diseases. Design.— First, we compared the staining intensity of both light chains between paraffin immunofluorescence and immunohistochemistry on a retrospective cohort of 28 cases of amyloidosis that have been previously typed. Then, we studied the role of paraffin immunofluorescence as an addition to our classical immunohistochemistry panel for amyloidosis typing. Results.— In the retrospective cohort, we found that paraffin immunofluorescence outperformed immunohistochemistry for light-chain detection. Then, in the prospective part of the study, we showed that the proportion of correctly classified cases increased from 50% to 71.9% with the adjunction of second-intention paraffin immunofluorescence to the immunohistochemistry procedure. Conclusions.— We therefore view paraffin immunofluorescence as a significant addition to the routine workflow for detection of light-chain–related diseases.

Immunoferritin localization of intracellular antigens in positively stained frozen sections

1978 ◽  
Vol 36 (2) ◽  
pp. 168-169
Author(s):  
K. T. Tokuyasu
Keyword(s):  
Surface Tension ◽  
Water Surface ◽  
Thin Layers ◽  
The Other ◽  
Positive Staining ◽  
Frozen Sections ◽  
The Past ◽  
Ultrathin Frozen Sections ◽  
Definition Of

During the past investigations of immunoferritin localization of intracellular antigens in ultrathin frozen sections, we found that the degree of negative staining required to delineate u1trastructural details was often too dense for the recognition of ferritin particles. The quality of positive staining of ultrathin frozen sections, on the other hand, has generally been far inferior to that attainable in conventional plastic embedded sections, particularly in the definition of membranes. As we discussed before, a main cause of this difficulty seemed to be the vulnerability of frozen sections to the damaging effects of air-water surface tension at the time of drying of the sections.Indeed, we found that the quality of positive staining is greatly improved when positively stained frozen sections are protected against the effects of surface tension by embedding them in thin layers of mechanically stable materials at the time of drying (unpublished).

Sign in / Sign up

Export Citation Format

Share Document