Journal of Histochemistry and Cytochemistry

The use of avidin-biotin interaction in immunoenzymatic techniques provides a simple and sensitive method to localize antigens in formalin-fixed tissues. Among the several staining procedures available, the ABC method, which involves an application of biotin-labeled secondary antibody followed by the addition of avidin-biotin-peroxidase complex, gives a superior result when compared to the unlabeled antibody method. The availability of biotin-binding sites in the complex is created by the incubation of a relative excess of avidin with biotin-labeled peroxidase. During formation of the complex, avidin acts as a bridge between biotin-labeled peroxidase molecules; and biotin-labeled peroxidase molecules, which contains several biotin moieties, serve as a link between the avidin molecules. Consequently, a "lattice" complex containing several peroxidase molecules is likely formed. Binding of this complex to the biotin moieties associated with secondary antibody results in a high staining intensity.

Các giai đoạn đầu tiên của quá trình hấp thụ peroxidase cây cải đuôi tiêm tĩnh mạch trong các ống thận gần của chuột đã được nghiên cứu bằng một kỹ thuật cytochemical cấu trúc siêu vi mới. Ở những con vật bị giết chỉ 90 giây sau khi tiêm, sản phẩm phản ứng được tìm thấy trên màng bờ chải và trong các chỗ hõm ống ở đỉnh. Từ các cấu trúc này, nó được vận chuyển đến các không bào đỉnh, nơi nó được tập trung dần để hình thành các giọt hấp thu protein. Phương pháp này, sử dụng 3,3'-diaminobenzidine làm chất nền có thể oxi hóa, cho phép định vị sắc nét và có độ nhạy cao. Hệ thống này rất thuận lợi trong việc nghiên cứu các giai đoạn đầu tiên của việc hấp thu protein qua ống thận, vì lượng nhỏ protein trên màng và trong ống cũng như các túi có thể dễ dàng phát hiện. Phương pháp này cũng cho thấy tiềm năng trong việc nghiên cứu sự vận chuyển protein ở nhiều loại tế bào và mô khác nhau.

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. s_{20, w}, 11.98 x 10^–13; d_{20, 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.

Tetramethyl benzidine (TMB) is a presumptively non-carcinogenic chromogen which yields a blue reaction-product at sites of horseradish peroxidase activity. Sixty-six distinct procedures were performed in rats and monkeys in order to determine the optimal incubation parameters for TMB. As a result, a procedure is recommended whose sensitivity greatly surpasses that of a previously described benzidine dihydrochloride method. Indeed, the sensitivity of this new method in demonstrating retrograde transport is markedly superior to that of the previously described benzidine dihydrochloride method. Furthermore, as a consequence of this enhanced sensitivity, many efferent connections of the injection site are also visualized. The injection site demonstrated by this TMB procedure is significantly larger than the one demonstrated when benzidine dihydrochloride or diaminobenzidine is used as a chromogen. Finally, this TMB procedure has been compared to two other TMB procedures and found to provide superior morphology and sensitivity.

A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure--the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method--gives excellent immunocytochemical labeling of tissue sections and cell smears, comparable in clarity and intensity to that achieved with immunoperoxidase labeling. If the enzyme label is developed with a naphthol salt as a coupling agent and Fast Red or hexazotized new fuchsin as a capture agent, a vivid red reaction product is obtained which is very easily detected by the human eye. For this reason the APAAP technique was found particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low numbers of antigen-bearing cells in a specimen (e.g., carcinoma cells in a malignant effusion). It was found possible to enhance the intensity of the APAAP labeling reaction substantially by repeating the second and third incubation steps (i.e., the unlabelled "bridge" antibody and APAAP complexes). The APAAP technique was superior to immunoperoxidase labeling for staining tissues rich in endogenous peroxidase, and could be used in conjunction with immunoperoxidase methods for double immunoenzymatic staining. The method was also applicable to the detection of antigenic molecules following their electrophoretic transfer from SDS-polyacrylamide gels to nitrocellulose sheets ("immunoblotting").

The reaction under mild conditions between formaldehyde and phenylalanine and phenylethylamine derivatives has been studied. When the amines included in a dried protein film were exposed to formaldehyde vapour a very intense green to yellow fluorescence was give only by those that as well as being primary amines also have hydroxyl groups at the 3 and 4 positions (3,4-dihydroxyphenylalanine, dopamine, noradrenaline). The 3-OH group seems to be esssential for the reaction. The catechol amines, which are secondary amines (adrenaline, epinine), gave a much weaker fluorescence that developed more slowly.

The results obtained on further examination of the reaction favour the view that the amines primarily condense with formaldehyde to 1,2,3,4-tetrahydroisoquinolines which are involved in a secondary reaction to become highly fluorescent and at the same time insoluble. This secondary reaction may be a binding to protein, and oxidation with the formation of double bonds in the heterocyclic ring, or both.

A new method of conjugating horseradish peroxidase with proteins was developed. The carbohydrate moiety of fluorodinitrobenzene-blocked peroxidase was oxidized with sodium periodate to form aldehyde groups. The peroxidase-aldehyde was then bound to free amino groups of proteins unidirectionally at high efficiencies. Peroxidase-labeled immunoglobulin retained its immunologic as well as enzymatic activities.

A group of apparently unrelated endocrine cells, some in endocrine glands, others in nonendocrine tissues, share a number of cytochemical and ultrastructural characteristics. These characteristics, from the initial letters of four of which the term APUD is derived, indicate the possession of a common metabolic pattern and common synthetic, storage and secretion mechanisms. It is postulated that the various characteristics reflect the production and storage of hormone precursor protein which has a predominantly random coil conformation. Several explanations are possible to account for the APUD characteristics but if the cells, in fact, share a common ancestor the only possible candidate is the neural crest cell.