An Improved Immunoperoxidase Technique Using Horseradish Peroxidase Avidin D

α-Fetoprotein (AFP) was successfully demonstrated in paraffin-embedded sections of human and rat fetal livers, by a multilayering immunohistochemical technique using the peroxidase-antiperoxidase system. Serial sections simultaneously subjected to immunofluorescence showed identical sites of localization in hepatocytic cytoplasm and sometimes in perivenous connective tissue. AFP-containing hepatocytes were located around efferent veins as well as randomly in the lobule. The immunoperoxidase technique has certain advantages over the immunofluorescence method which would justify the former's application in studies on the dynamics of AFP synthesis.

Download Full-text

A double-label pre-embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethylbenzidine as markers.

Journal of Histochemistry & Cytochemistry ◽

10.1177/37.8.2666511 ◽

1989 ◽

Vol 37 (8) ◽

pp. 1283-1289 ◽

Cited By ~ 55

Author(s):

R B Norgren ◽

M N Lehman

Keyword(s):

Electron Microscopy ◽

Luteinizing Hormone ◽

Horseradish Peroxidase ◽

Preoptic Area ◽

Immunoperoxidase Technique ◽

Tract Tracing ◽

Electron Microscopic ◽

Luteinizing Hormone Releasing Hormone ◽

Double Label ◽

Better Than

Techniques for correlative double-label immunocytochemistry (ICC) at light and electron microscopic (EM) level are useful for determining the neurotransmitter phenotype of inputs onto immunocytochemically identified neurons. Tetramethylbenzidine (TMB) has been used as a chromogen at the EM level for horseradish peroxidase tract tracing. We have found that TMB, in combination with diaminobenzidine (DAB), can be used in a double-label immunocytochemical protocol to examine neuropeptide Y inputs onto luteinizing hormone-releasing hormone cells in the sheep preoptic area. At both light and EM levels, TMB reaction product is visibly distinct from DAB reaction product. The ultrastructural preservation we have been able to obtain with our technique is better than that obtained with techniques that use TMB at a lower pH. Furthermore, this technique allows the demonstration of synaptic contacts between neurochemically identified terminals and cells with different neurotransmitter phenotypes.

Download Full-text

L'identification du virus de l'influenza à l'aide de l'immunoperoxydase

Canadian Journal of Microbiology ◽

10.1139/m73-021 ◽

1973 ◽

Vol 19 (1) ◽

pp. 147-149 ◽

Cited By ~ 3

Author(s):

R. Dobardzic ◽

A. Boudreault ◽

V. Pavilanis

Keyword(s):

Influenza Virus ◽

Chick Embryo ◽

Horseradish Peroxidase ◽

Coupling Agent ◽

Light Microscope ◽

Viral Antigen ◽

Immunoperoxidase Technique ◽

Kidney Cells ◽

Virus Infections ◽

Specific Antibodies

The specific antibodies anti-influenza virus A2/Aichi/68 were combined immunologically with the virus purified by zonal centrifugation, then dissociated by lowering the pH and conjugated with horseradish peroxidase using glutaraldehyde as the coupling agent. With so labeled antibodies it was possible to detect the viral antigen in infected chick embryo kidney cells with the light microscope. This work suggests that the immunoperoxidase technique could be useful for early rhinocytologic diagnosis of influenza virus infections.

Download Full-text

Effects of Vincristine on Endothelial Microtubules in the Spinal Cord

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090270 ◽

1976 ◽

Vol 34 ◽

pp. 58-59

Author(s):

John L. Beggs ◽

John D. Waggener ◽

Wanda Miller

Keyword(s):

Spinal Cord ◽

Biological Activity ◽

Horseradish Peroxidase ◽

Transport Mechanism ◽

Vasogenic Edema ◽

Vesicular Transport ◽

Close Proximity

Microtubules (MT) are versatile organelles participating in a wide variety of biological activity. MT involvement in the movement and transport of cytoplasmic components has been well documented. In the course of our study on trauma-induced vasogenic edema in the spinal cord we have concluded that endothelial vesicles contribute to the edema process. Using horseradish peroxidase as a vascular tracer, labeled endothelial vesicles were present in all situations expected if a vesicular transport mechanism was in operation. Frequently,labeled vesicles coalesced to form channels that appeared to traverse the endothelium. The presence of MT in close proximity to labeled vesicles sugg ested that MT may play a role in vesicular activity.

Download Full-text

Comparative strengths and weaknesses of peroxidase and colloidal gold in pre- and post-embedding immunolabeling techniques

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100129279 ◽

1987 ◽

Vol 45 ◽

pp. 1002-1005

Author(s):

D. R. Abrahamson ◽

P. L. St.John ◽

E. W. Perry

Keyword(s):

Electron Microscopy ◽

Horseradish Peroxidase ◽

Light Microscopy ◽

Colloidal Gold ◽

Light Microscope ◽

Ultrastructural Localization ◽

The Other ◽

Quantitative Studies ◽

Dense Suspension ◽

Antigen Localization

Antibodies coupled to tracers for electron microscopy have been instrumental in the ultrastructural localization of antigens within cells and tissues. Among the most popular tracers are horseradish peroxidase (HRP), an enzyme that yields an osmiophilic reaction product, and colloidal gold, an electron dense suspension of particles. Some advantages of IgG-HRP conjugates are that they are readily synthesized, relatively small, and the immunolabeling obtained in a given experiment can be evaluated in the light microscope. In contrast, colloidal gold conjugates are available in different size ranges and multiple labeling as well as quantitative studies can therefore be undertaken through particle counting. On the other hand, gold conjugates are generally larger than those of HRP but usually can not be visualized with light microscopy. Concern has been raised, however, that HRP reaction product, which is exquisitely sensitive when generated properly, may in some cases distribute to sites distant from the original binding of the conjugate and therefore result in spurious antigen localization.

Download Full-text

Unlabeled Antibody Immunocytochemistry Applied to Murine Mammary Tumor Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115572 ◽

1975 ◽

Vol 33 ◽

pp. 390-391

Author(s):

Wm. J. Arnold ◽

J. Russo ◽

H. D. Soule ◽

M. A. Rich

Keyword(s):

Horseradish Peroxidase ◽

Mammary Tumor ◽

Covalent Binding ◽

Petri Dish ◽

Gamma Chain ◽

Mammary Tumor Virus ◽

Mammary Tumor Cells ◽

Murine Mammary Tumor ◽

Tumor Virus ◽

Unlabeled Antibody

Our studies of mammary tumor virus have included the application of the unlabeled antibody enzyme method of Sternberger to mammary tumor derived mouse cells in culture and observation with an electron microscope. The method avoids the extravagance of covalent binding of indicator molecules (horseradish peroxidase) with precious antibody locator molecules by relying instead upon specific antibody-antigen linkages. Our reagents included: Primary Antibody, rabbit anti-murine mammary tumor virus (MuMTV) which was antiserum 113 AV-2; Secondary Antibody, goat anti-rabbit IgG gamma chain (Cappel Laboratories); andthe Indicator, rabbit anti-horseradish peroxidase - horseradish peroxidase complex (PAP) (Cappel Labs.). Dilutions and washes were made in 0.05 M Tris 0.15 M saline buffered to pH 7.4. Cell monolayers, after light fixation in glutaraldehyde, were incubated in place by a protocol adapted from Sternberger and Graham and Karnovsky, then embedded by our usual method for monolayers. Reagents were confined to specific areas by neoprene 0-rings (Parker Seal Co.) reducing the amount of reagent needed to 50 microliters, 1/6th of that required to wet a 35 mm petri dish.

Download Full-text