Journal of Histochemistry and Cytochemistry

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.

The avian song control system is an excellent model in which to study the effects of gonadal steroid hormones on neural and behavioral plasticity. Several of the brain regions that control song behavior concentrate androgens and/or estrogens. Investigations of the distribution and regulation of androgen receptors have been limited by the lack of a reliable immunocytochemical method to detect androgen receptors in the songbird brain. We describe a protocol by which the PG-21 polygonal antibody to the rat androgen receptor can be used to label androgen receptor-containing cells in the songbird brain. By treating songbirds of several species with testosterone 90 min before sacrifice and by using relatively low concentrations (0.5 0.75 microg/ml) of PG-21 antibody to reduce nonspecific background staining, we were able to obtain strong specific labeling of cell nuclei in androgen-sensitive brain regions. This technique will facilitate the study of the role of androgens in mediating neural plasticity in the avian brain. Testosterone pretreatment may also facilitate the use of this antibody to label androgen receptors in tissues from a wide array of nonmammalian species.

We developed a method involving air-drying of a rubber suspension after fixation in glutaraldehyde-tannic acid and postfixation in osmium tetroxide for SEM observation. For TEM immunolabeling the suspension was air-dried after osmium-only fixation. Whereas conventional methods failed to satisfactorily stabilize rubber particles, the methods described here proved successful in preserving their integrity.

Một phương pháp được mô tả cho việc cố định các khối mô để sử dụng trong các nghiên cứu sử dụng huỳnh quang miễn dịch. Phương pháp này bao gồm việc cố định các khối mỏng trong ethanol 95% và tiến hành khử nước và làm trong ở nhiệt độ tủ lạnh (4°C). Sau đó, việc thấm paraffin và cắt lát bằng phương pháp vi thể tiêu chuẩn trở nên dễ dàng.

Phương pháp này tạo ra các chế phẩm có độ chính xác cao hơn về mặt mô học trong việc xác định vị trí kháng nguyên hoặc kháng thể so với các chế phẩm từ mô đông lạnh; và với kháng thể thỏ và albumin huyết thanh bò, độ nhạy của phát hiện được cải thiện. Albumin huyết thanh bò có thể được tìm thấy trong thời gian dài hơn sau khi tiêm so với các lát cắt đông lạnh.

Các kháng nguyên khác mà quy trình này đã chứng minh là đạt yêu cầu bao gồm gamma globulin bò, ferritin ngựa, virus cúm A, giải độc tố bạch hầu và uốn ván. Ovalbumin gà bị suy giảm.

Các kháng nguyên mới hoặc kháng thể mới nên được thử nghiệm trước khi thực hiện theo phương pháp này.

Adhesive glycoproteins in the bone matrix are of critical importance for cell anchorage, proliferation, migration, differentiation, and regulation of bone metabolism. The localization of the adhesive glycoprotein vitronectin (Vn) in murine bone tissue was evaluated by immunohistochemical staining. Vitronectin was present throughout the mineralized bone matrix of cancellous and cortical bone, whereas cartilage was devoid of Vn staining. To exclude the possibility that the positive Vn staining resulted from plasma Vn in blood vessels within the bone sections, adjacent tissue sections were stained with antibodies to fibrinogen, and abundant plasma protein. Fibrinogen immunoreactivity was confined to blood vessels in the bone marrow and Haversian system, whereas the mineralized bone matrix was devoid of staining. The presence of Vn in murine bones was confirmed by sequential extraction, followed by fractionation of the resulting polypeptides by gel electrophoresis and immunoblotting analysis. Hydroxyapatite affinity chromatography raises the possibility that mineral interactions, at least in part, mediate the incorporation of Vn into the bone matrix. These results indicate that Vn is a specific component of bone tissue and raise the possibility that Vn is involved in regulation of bone metabolism.

A newly defined endothelial cell permeability structure, termed the Vesiculo–Vacuolar organelle (VVO), has been identified in the microvasculature that accompanies tumors, in venules associated with allergic inflammation, and in the endothelia of normal venules. This organelle provides the major route of extravasation of macromolecules at sites of increased vascular permeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), serotonin, and histamine in animal models. Continuity of these large sessile structures between the vascular lumen and the extracellular space has been demonstrated in kinetic studies with ultrastructural electron-dense tracers, by direct observation of tilted electron micrographs, and by ultrathin serial sections with three-dimensional computer reconstructions. Ultrastructural enzyme-affinity cytochemical and immunocytochemical studies have identified histamine and VPF/VEGF bound to VVOs in vivo in animal models in which these mediators of permeability are released from mast cells and tumor cells, respectively. The high-affinity receptor for VPF/VEGF, VEGFR-2, was localized to VVOs and their substructural components by pre-embedding ultrastructural immunonanogold and immunoperoxidase techniques. Similar methods were used to localize caveolin and vesicle-associated membrane protein (VAMP) to VVOs and caveolae, indicating a possible commonality of formation and function of VVOs to caveolae.

We describe a novel histochemical procedure for simultaneous detection of mRNA expression by in situ hybridization (ISH) and DNA synthesis on cells that are pulse-labeled with bromodeoxyuridine (BrdU) by immunohistochemistry (ICC). Pregnant rats were injected with BrdU at embryonic Day 20 and the olfactory bulbs of their pups were collected daily. The expression of calmodulin (CaM) mRNA was analyzed by ISH with an anti-sense digoxigenin-labeled riboprobe and BrdU incorporation by indirect ICC. Starting 5 days after BrdU injection, a few tufted and granular neurons of the olfactory bulb were observed to be double labeled for CaM mRNA and BrdU. To study the olfactory neuroepithelium, adult animals were injected with BrdU, sacrificed after 30 days, and the nasal mucosa dissected and decalcified. The co-expression of CaM mRNA and BrdU incorporation was then analyzed in the olfactory neuroepithelium: BrdU-positive primary olfactory neurons were also CaM mRNA positive. The combination of ISH and ICC on the same section resulted in improved BrdU staining with respect to both increased intensity and reduced background levels. The procedure described here can be applied to a variety of problems in developmental biology and is of potential value for correlating the timing of specific mRNA expression with the birth date of a cell type of interest.

A procedure is presented which modifies the Sternberger peroxidase--antiperoxidase (PAP) technique in order to visualize additional amounts of immunodeposits representing the antigen substance (SP) in 5-micrometer paraffin tissue sections of rat spinal cord. For increased sensitivity, the new procedure utilizes a "double bridge" and diaminobenzidine in low pH buffer. The modifications have made possible the visualization of immunoreactive beaded processes and punctate bodies, which were then traced to determine patterns of SP circuitry. Using the modified PAP procedure, the greatest number of immunoreactive processes appeared in the dorsal horn, where some punctate bodies and varicose processes could be seen adjacent to the myelinated afferent fiber bundles that penetrate the substantia gelatinosa as dorsal root collaterals. Additional immunoreactive processes and punctate bodies coursed through the myelinated afferent fiber bundles that penetrate the dorsolateral white matter, and extend into the intermediolateral gray region. Substance P was also identified within immunoreactive processes found in Rexed's laminae V and VI, as well as the central canal region, the dorsal gray commissure, and the ventral gray and white commissures. Since the modifications improved the visualization of SP-containing processes in sparsely populated regions of the spinal cord, especially the ventral horn, they may be useful in demonstrating other antigens that normally occur in small quantities within tissues.