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.

Immunohistochemical Localization of ACTH and Prolactin in the Pituitary Gland of Adult Migratory Sockeye Salmon (Oncorhynchus nerka)

1969 ◽  
Vol 26 (7) ◽  
pp. 1837-1846 ◽  
Author(s):  
B. A. McKeown ◽  
A. P. van Overbeeke
Keyword(s):  
Sockeye Salmon ◽  
Fluorescein Isothiocyanate ◽  
Cell Types ◽  
Pituitary Hormones ◽  
Immunohistochemical Localization ◽  
Granule Density ◽  
Pituitary Glands ◽  
Antibody Complex ◽  
Ovine Prolactin ◽  
Antigen Antibody

Antibodies to porcine adrenocorticotrophic hormone (ACTH), Synacthen (synthetic corticotrophin, Ciba), and ovine prolactin were prepared in rabbits and the antisera were tested for specificity against several pituitary hormones. The gamma-globulin fractions of the antisera were conjugated with fluorescein isothiocyanate and the labelled antibodies were "purified" by column chromatography.Fresh-frozen sections of pituitary glands of adult migratory sockeye salmon were incubated with the antibody solutions and examined with a fluorescence microscope. The resulting antigen–antibody complex could be localized by re-photographing the same or alternate sections after fixation and staining. Anti-ACTH and anti-Synacthen appeared to be bound specifically by the epsilon cells, whereas anti-prolactin reacted with the eta cells of the rostral pars distalis. Pituitary glands collected at various stations along the migratory route, including one seawater sample, showed the same reactivity. Other glands were prepared for histological examination. Microspectrophotometric analysis of cell types showed that the granule density of the ACTH cells increased gradually during the later part of migration. In the prolactin cells, no change in granulation could be detected during entrance into the river or subsequent spawning migration.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF CONTRACTILE PROTEIN (THROMBOSTHENIN) IN HUMAN PLATELETS USING AN UNLABELED ANTIBODY-PEROXIDASE STAINING TECHNIQUE

1971 ◽  
Vol 19 (9) ◽  
pp. 540-550 ◽  
Author(s):  
FRANCOIS M. BOOYSE ◽  
DOROTHEA ZSCHOCKE ◽  
MAX E. RAFELSON ◽  
LUDWIG A. STERNBERGER
Keyword(s):  
Horseradish Peroxidase ◽  
Staining Technique ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Contractile Protein ◽  
Intact Cells ◽  
Proteinaceous Material ◽  
Antibody Staining ◽  
Ultrathin Sections ◽  
Unlabeled Antibody

Soluble horseradish peroxidase-antihorseradish peroxidase (rabbit) complex was used to localize the actomyosin-like contractile protein, thrombosthenin, in both intact human platelets (Epon-embedded) and ultrathin sections (methacrylate-embedded). Antibody staining of ultrathin sections showed the presence of membrane-associated and cytoplasmic thrombosthenin. Antibody staining of intact cells showed that membrane-associated thrombosthenin was localized, at least in part, in the exterior, "fluffy" coat of the platelet. Even after prolonged fixation and/or incubation with the various antisera, we were unable to demonstrate antibody penetration of intact, fixed platelets. Brief treatment with Pronase removed the surface-localized proteinaceous material and completely abolished all antibody staining.

scholarly journals THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOHISTOCHEMISTRY PREPARATION AND PROPERTIES OF SOLUBLE ANTIGEN-ANTIBODY COMPLEX (HORSERADISH PEROXIDASE-ANTIHORSERADISH PEROXIDASE) AND ITS USE IN IDENTIFICATION OF SPIROCHETES

1970 ◽  
Vol 18 (5) ◽  
pp. 315-333 ◽  
Author(s):  
LUDWIG A. STERNBERGER ◽  
PAUL H. HARDY ◽  
JOHN J. CUCULIS ◽  
HOWARD G. MEYER
Keyword(s):  
Horseradish Peroxidase ◽  
Rabbit Antiserum ◽  
Antibody Fragment ◽  
Soluble Antigen ◽  
Simple Method ◽  
Antibody Complex ◽  
High Yields ◽  
Antigen Antibody ◽  
Unlabeled Antibody ◽  
Specific Rabbit

Antigen was identified histochemically without the use of labeled antibodies by the sequential application of (a) specific rabbit antiserum, (b) sheep antiserum to rabbit immunoglobulin G, (c) specifically purified, soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP), (d) 3,3'-diaminobenzidine and hydrogen peroxide and (e) osmium tetroxide. A simple method for preparation of high yields of PAP consisted of precipitation of antibody from specific rabbit antiserum with horseradish peroxidase (PO) at equivalence, solubilization of the washed precipitate with excess PO at pH 2.3, 1°C, followed by immediate neutralization and separation of PAP from PO by half-saturation with ammonium sulfate. The ratio of PO to anti-PO in PAP was 3:2 irrespective of the source of antiserum. PAP was heterogeneous on electrophoresis, homogeneous on sedimentation, diffusion and electron microscopy and consisted of pentagons with diameters of 205 Å. s20, w, 11.98 x 10–13; d20, w, 2.48 x 10–7; molecular weight by sedimentation velocity, 429,000, and equilibrium, 413,000. Sensitivity and specificity of immunohistochemical staining of spirochetes was about 100- to 1000-fold that of immunofluorescence. The unexpected ratio of PO to anti-PO is presumed to be due to stabilization by the pentagonal shape in which three corners are suspected to be PO and two antibody fragment Fc.

Visualization of Antibody Transport in Rat Visceral Yolk-Sac Placenta

1982 ◽  
Vol 40 ◽  
pp. 246-247
Author(s):  
William P. Jollie
Keyword(s):  
Phosphate Buffer ◽  
Yolk Sac ◽  
Female Rats ◽  
Thin Sections ◽  
Visceral Yolk Sac ◽  
Antibody Complex ◽  
Cytochemical Reaction ◽  
Hind Foot ◽  
Antigen Antibody ◽  
Yolk Sac Placenta

A technique has been developed for visualizing antibody against horseradish peroxidase (HRP) in rat visceral yolk sac, the placental membrane across which passive immunity previously has been shown to be transferred from mother to young just prior to birth. Female rats were immunized by injecting both hind foot pads with 1 mg HRP emulsified in complete Freund's adjuvant. They were given a booster of 0.5mg HRP in 0.1 ml normal saline i.v. after one week, then bred and autopsied at selected stages of pregnancy, viz., 12, 1 7 and 22 days post coitum, receiving a second booster, injected as above, five days before autopsy. Yolk sacs were removed surgically and fixed immediately in 2% paraformaldehye, 1% glutaraldehye in 0.1 M phosphate buffer with 0.01% CaCl2 at pH 7.4, room temperature, for 3 hr, rinsed 3X in 0.1 M phosphate buffer plus 5% sucrose, then exposed to 1 mg HRP in 1 ml 0.1 M phosphate buffer at pH 7.4 for 1 hr. They were refixed in aldehydes, as above, for 1 5 min (to assure binding of antigen-antibody complex). Following buffer washes, the tissues were incubated in 3 mg diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 0.05 M Tris-HCl buffer for 30 min. After brief buffer washes, they were postfixed in 2% OsO4. in phosphate buffer at pH 7.4, 4°C for 2 hr, dehydrated through a graded series of ethanols, and embedded in Durcupan. Thin sections were observed and photographed without contrast-enhancement with heavy metals. Cytochemical reaction product marked the site of HRP (i.e., antigen) which, in turn, was present only where it was bound with anti-HRP antibody.

Ultrastructural localization of neuropeptides in the rat brain

1978 ◽  
Vol 36 (2) ◽  
pp. 612-613
Author(s):  
F.W. Van Leeuwen
Keyword(s):  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Serial Sections ◽  
Microscopical Level ◽  
Electron Microscopical Level ◽  
Enzyme Method ◽  
Perfusion Fixation ◽  
Electron Microscopical ◽  
Unlabeled Antibody ◽  
Peroxidase Conjugate

In order to obtain specific and optimal ultrastructural localization of vasopressin and oxytocin in the hypothalamo-neurohypophyseal system of the rat, 2 staining procedures and several tissue treatments were evaluated using neurohypophyseal tissue. It appeared from these studies that post-embedding staining with the unlabeled antibody enzyme method developed by Sternberger allows greater dilution of the first antibody (anti-vasopressin, 1:4800) than the indirect procedure (1:320) using a peroxidase conjugate as second antibody. Immersion fixation with 4% formalin during 24 hours gave better results (general ultrastructure, immunoreactivity) than those obtained by perfusion fixation with 2.5% glutaraldehyde-1% paraformaldehyde or freeze substitution.Since no reliable specificity tests were performed at the electron microscopical level, tests were developed for antibodies against both vasopressin and oxytocin. For anti-vasopressin plasma neural lobes of homozygous Brattleboro rats, that are lacking vasopressin by a genet- ical defect, were used. For antibodies against both hormones serial sections were used that were alternately incubated with the antibodies.

INHIBITION OF LIPOLYTIC ACTIVITY OF SYNTHETIC β1–24 AND β1–39 CORTICOTROPHIN BY ANTI-ACTH ANTISERUM

1966 ◽  
Vol 51 (1) ◽  
pp. 88-94 ◽  
Author(s):  
A. Villanueva ◽  
S. J. H. Ashcroft ◽  
J. P. Felber
Keyword(s):  
Guinea Pig ◽  
Lipolytic Activity ◽  
Peptide Hormones ◽  
Fat Pad ◽  
Double Antibody ◽  
Epididymal Fat ◽  
Antibody Complex ◽  
Radio Immunoassay ◽  
Antigen Antibody

ABSTRACT The synthetic ACTH peptides β1–39 and β1–24 stimulated lipolysis as determined by the rat epididymal fat pad in vitro. The stimulating effect of these peptides was diminished by prior incubation of the peptides with antibodies produced by the guinea-pig against ACTH. The stimulating effect of these hormones was also diminished by the double antibody system used in the radio-immunoassay of ACTH and other peptide hormones, in which incubation with antiserum is followed by precipitation of the antigen-antibody complex by rabbit anti-guinea-pig-γ-globulin.

IDENTIFICATION OF LATS AS AN ANTIGEN-ANTIBODY COMPLEX

1973 ◽  
Vol 71 (4_Suppl) ◽  
pp. S11
Author(s):  
K. Schemmel ◽  
L. Weisbecker ◽  
H. Norden ◽  
V. Mokmol ◽  
V. Becker ◽  
...  
Keyword(s):  
Antibody Complex ◽  
Antigen Antibody

Immunohistochemical and ultrastructural localization of Schistosoma mansoni soluble egg antigens processed by the infected host

Parasitology ◽  
1996 ◽  
Vol 112 (6) ◽  
pp. 537-543 ◽  
Author(s):  
J. J. P. M. Bogers ◽  
H. A. M. Nibbeling ◽  
A. M. Deelder ◽  
E. A. E. Van Marck
Keyword(s):  
Schistosoma Mansoni ◽  
Indirect Evidence ◽  
Ultrastructural Localization ◽  
Carbohydrate Epitopes ◽  
Eosinophilic Granulocytes ◽  
Antigenic Material ◽  
Egg Shells ◽  
Secondary Lysosomes ◽  
Antigen Antibody ◽  
Egg Antigen

SUMMARYThe detection of egg-derived antigens in the serum and urine of Schistosoma mansoni-infected individuals and experimental animals would provide an alternative method to assess the tissue egg burden. The detected levels are, however, not only a function of the amounts of antigen produced, but also of the processing or clearance by the host. In the present study the immunolocalization pattern of antigens using 2 recently described monoclonal antibodies to repetitive carbohydrate epitopes of S. mansoni soluble egg antigen (114–5B1–A and 114–4D12–A) in various organs of the host was investigated. In the liver strong immunoreactivity could be detected around the entrapped eggs and in egg-shells, as well as in Kupffer cells accumulating both antigen and schistosomal pigment. In the spleen, immunohistochemistry revealed antigen in the plasma as well as in secondary lysosomes of macrophages. Strong labelling was found in the vesicles of the eosinophilic granulocytes: indirect evidence perhaps for the presence of antigen–antibody complexes. In conclusion, the secreted egg antigens were sequestered in the reticulo-endothelial macrophages of the liver and the spleen as already partly described for worm-derived antigens. The presence of large quantities of antigenic material in the spleen could suggest an important role of this organ in the clearance of antigen and might even provide an additional explanation for the hepatosplenomegaly mainly present in S. mansoni-infected children.

scholarly journals Solubility of cytoskeletal proteins in immunohistochemistry and the influence of fixation.

1993 ◽  
Vol 41 (4) ◽  
pp. 609-616 ◽  
Author(s):  
B M Riederer ◽  
R Porchet ◽  
R A Marugg ◽  
L I Binder
Keyword(s):  
Protein Solubility ◽  
Immunohistochemical Localization ◽  
Influential Factors ◽  
Cytoskeletal Proteins ◽  
Fixed Tissue ◽  
Aldehyde Fixation ◽  
Cat Brain ◽  
Spectrin Cytoskeleton ◽  
Cryostat Sections

For accurate and quantitative immunohistochemical localization of antigens it is crucial to know the solubility of tissue proteins and their degree of loss during processing. In this study we focused on the solubility of several cytoskeletal proteins in cat brain tissue at various ages and their loss during immunohistochemical procedures. We further examined whether fixation affected either solubility or immunocytochemical detectability of several cytoskeletal proteins. An assay was designed to measure the solubility of cytoskeletal proteins in cryostat sections. Quantity and quality of proteins lost or remaining in tissue were measured and analyzed by electrophoresis and immunoblots. Most microtubule proteins were found to be soluble in unfixed and alcohol fixed tissues. Furthermore, the microtubule proteins remaining in the tissue had a changed cellular distribution. In contrast, brain spectrin and all three neurofilament subunits were insoluble and remained in the tissue, allowing their immunocytochemical localization in alcohol-fixed tissue. Synapsin I, a protein associated with the spectrin cytoskeleton, was soluble, and aldehyde fixation is advised for its immunohistochemical localization. With aldehyde fixation, the immunoreactivity of some antibodies against neurofilament proteins was reduced in axons unveiling novel immunogenic sites in nuclei that may represent artifacts of fixation. In conclusion, protein solubility and the effects of fixation are influential factors in cytoskeletal immunohistochemistry, and should be considered before assessments for a quantitative distribution are made.

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