A Rapid Silver Staining and Destaining Technique for the Nucleolus Organizer Region

Silver staining of nucleolar organizing regions (NOR) is common, but a standard protocol is lacking. A modification of a rapid silver nitrate staining technique for NORs is presented here. Advantages of the modified technique include reliability, speed, cost and the fact that it can be carried out in the light.     

Microwave Enhanced Staining for Plant Virus Inclusions

Plant virus inclusion bodies can be stained specifically with established staining methods for light microscopy. The procedure can be augmented by a short microwave treatment to provide better staining intensity and reduced staining time. The method is useful for preliminary sampling prior to collection for electron microscopy and for plant pathologists, plant breeders, and diagnosticians as a rapid means of plant virus characterization.     

An Automated Technique for Double Staining Rat and Rabbit Fetal Skeletal Specimens to Differentiate Bone and Cartilage

Assessment of chemicals for their potential to cause developmental toxicity must include evaluation of the development of the fetal skeleton. The method described here is an improved and fully automated double staining method using alizarin red S to stain bone and alcian blue to stain cartilage. The method was developed on the enclosed Shandon PathcentreTM, and the quality of specimens reported here will be reproduced only if carried out on a similar processor under the same environmental conditions. The staining, maceration and clearing process takes approximately 6 days. The personnel time, however, is minimal since solutions are changed automatically and the fetuses are not examined or removed from the processor until the procedure is completed. Upon completion of processing, the bone and cartilage assessment of the specimens can be carried out immediately if required. Full evaluation of skeletal development in both the rat and the rabbit is necessary to meet the requirements of safety assessment studies. This method allows this to be accomplished on a large scale with consistently clear specimens and in a realistic time.     

微卫星DNA聚丙烯酰胺凝胶电泳(PAGE)银染法的改良

为了筛选和检测绵羊种群中具有较好表现的微卫星多态性,采用Touch-down PCR方法扩增绵羊基因组上的微卫星DNA序列后,经聚丙烯酰胺凝胶电泳分离,采用改良的银染法对微卫星位点进行多态性检测,染色结果与常规银染法相比,该方法具有灵敏度高、背景浅、污染小、条带清晰等突出特点,能有效地控制染色背景,显著提高检测分辨率,整个染色过程所需时间短,具有广泛的推广价值。     

Staining Plant Cells with Silver. I. The Salt-Nylon Technique

A technique is described for selectively silver staining nucleoli, active nucleolus organizers, nucleolar material attached to chromosomes, kinetochores, synaptonemal complexes, and chromosome cores in plant cells. The technique, called salt-nylon silver staining, involves spreading cells on glass slides, treating the cells with a solution of saline sodium citrate, and incubating the cells in a silver nitrate solution covered with nylon screen. Selected variables important for achieving reliable silver staining are considered.     

Immunohistochemical Double Staining with Immunogold-Silver and Alkaline Phosphatase to Identify Nuclear Markers of Cellular Proliferation

To facilitate cell kinetics studies of brain tumors labeled with thymidine analogs, we developed a new method to identify nuclei labeled sequentially with bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR) by double staining with immunogold-silver and alkaline phosphatase. Patients received an intraoperative infusion of BUdR: excised tumor specimens were immediately labeled with IUdR in vitro. fixed with 70% alcohol, embedded in paraffin, and cut into 6 pm sections. The sections were incubated first with BR-3. a monoclonal antibody that recognizes only BUdR, and then with IU-4. a monoclonal antibody that recognizes both BUdR and IUdR: sections were counterstained with hematoxylin to identify unlabeled nuclei. Nuclei labeled only with IUdR stained red, whereas those labeled with BUdR or with both BUdR and IUdR stained black against a red background: unlabeled nuclei stained blue. This method was the most efficient differential staining technique to identify nuclei labeled only with IUdR and those labeled with BUdR among unlabeled nuclei.     

A Simple and Rapid Staining Technique for Plastic Embedded Cartilage and Bone

In this report we describe a simple and rapid staining technique for cartilage and bone embedded in Araldite. Semitbin sections of embryonic vertebrae obtained from 15 to 17 day mouse fetuses were stained using an aqueous solution 0.25% with respect to methylene blue, 0.25% with respect to azure A, and 0.5% with respect to Na₂ CO₃, then counterstained with 1% aqueous pararosaniline chloride (MAP). Results were compared with toluidine blue stained sections. MAP permitted good discrimination of developmental stages of both cells and extracellular matrix within vertebral ossification centers during endochondral ossification. The technique is simple, rapid and applicable to plastic embedded sections, and can be used prior to ultrastructural examination.     

Microwave Irradiation for Rapid and Enhanced lmmunohistochemical Staining: Application to Skin Antigens

We describe a method in which microwave irradiation is used to reduce substantially the incubation time for immunoperoxidase staining of antigens in cryostat sections of pso-riatic skin. An incubation time of 5-9 min irradiation at 80 W generated similar or better staining intensity for all antibodies used compared to the standard methods using 30-60 min incubation at room temperature. Although we found that microwave irradiation could be used with all antibodies tested, independent of whether they recognized extracellular, membrane or cytoplasmic antigens in skin, the conditions needed to be optimized for each antibody.     

一种改良的肌细胞骨架染色方法

为了观察肌细胞骨架,对传统考马斯亮蓝染色法进行改良,并与免疫荧光染色法进行了比较。培养的血管平滑肌细胞先用多聚甲醛预固定后再进行考马斯亮蓝染色,可使细胞骨架非常清晰的显色,解决了传统考马斯亮蓝染色易使肌细胞变形、脱片的问题,其效果与免疫荧光染色相近。因此,多聚甲醛预固定．考马斯亮蓝染色法是一种适于肌细胞骨架染色的简便方法。     

A Double Staining Technique using 5-Bromo,4-chloro-3-indolyl-β-D- galactopyranoside (X-gal) and Immunoperoxidase in Whole Drosophila Embryos

We have developed a double staining method using 5-bromo,4-chloro-3-indolyl-β-D-galactopyranoside X-gal and immunoperoxidase for whole Drosophila embryos. The dechorionated embryos are fixed in heptane saturated with 4% formaldehyde, then in heptane and 50% methanol. Fixed embryos are devitellinized with a tungsten needle and processed for immunoperoxidase staining immediately prior to peroxidase color development. The embryos are stained with X-gal, then peroxidase staining is resumed. This procedure enables us to observe cells stained with both X-gal and a specific antibody in whole embryos.     

Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

A Formalin-Wright Staining Technique for Avian Blood Cells

A rather concentrated alcoholic staining solution, an aqueous formalin-containing diluent, and a mixture of ethyl ether and absolute methyl alcohol are required. Formulas: A. Wright's stain (Harleco, Cert. No. LWr-52 was used), 3.3 gm; methyl alcohol, 500 ml. B. Formaldehyde solution 40% USP (Fisher's used), 0.25 ml; distilled water, 500 ml with its pH adjusted to 6.8 by addition of either 0.25% Na₂CO₂ or 0.25% HCl, as needed. C. A I:I mixture of ethyl ether and absolute methyl alcohol. Procedure: Prepare thin smears of normal or pathological avian blood, air dry, place the slides on a drying rack, cover with solution A, and let stand for about 8 min. Dilute the stain by dropping on a volume of B estimated to be equal to the volume of the partially evaporated stain, and let stand for 2-5 min, or until the surface is well covered by a metallic sheen. Wash with distilled water adjusted to pH 6.8 with the 0.25% Na₂CO₂ solution or 0.25% HCl. Dry the preparations quickly by blotting with filter paper. Differentiate and adjust the color intensities by dipping 6-10 times into C. Check the results microscopically and differentiate further if the colors are not properly balanced. Dry, uncovered preparations may be examined under oil; or, a cover glass can be applied with balsam or a synthetic resin for permanent mount. Results are similar to those described in textbooks, but have been more consistent than those obtained with other techniques for blood cells of chicken, pheasants, American and Indian partridge, quail, pigeon, turkey, goose, canary, and the Himalayan snow partridge.     

莱茵衣藻和斜生栅藻蛋白核的染色方法优化

蛋白核（pyrenoid）作为真核藻类重要的、相对独立的固碳结构,在CO2高效浓缩和固定过程中发挥中重要作用,可为开发高效生物固碳技术提供理论支持。染色法是快速观察蛋白核的最佳方法之一,有助于深入研究蛋白核的结构和功能,但目前对染色法仍缺乏系统研究。本文以较易染色的莱茵衣藻（Chlamydomonas reinhardtii）和较难染色的斜生栅藻（Scenedesmus obliquus）为材料,探讨了碘染和溴酚蓝（BPB）染色观察蛋白核的方法。结果表明,碘染法最适用于蛋白核外周淀粉观察,最适染色液浓度和时间分别是0．1％～0．2％（w／v）和5min。热激预处理和溴酚蓝染色相结合的蛋白核染色效果更好,优化条件为：70℃水浴热激40s,0．05％（w／v）BPB染色15min。本方法为研究微藻蛋白核提供了一种快速有效的直接观察方法。     

A Rapid Silver-Free Method for Staining the Myenteric Plexus

A method is presented for the relatively rapid demonstration of the myenteric plexus. Saturated Sudan black B in 70% ethanol followed by 0.01% aqueous buffered thionin were used on intestinal peels (whole-mounts) to stain myelinated and unmyelinated fibers and neuron cell bodies, respectively. In contrast to accepted silver methods, these two kinds of fibers were distinguished clearly; Schwann cell nuclei and nodes of Ranvier were visible. Preparations had the following attributes: relatively low optical density coupled with high visual contrast, freedom from metallic “mirroring,” low background staining of subjacent muscle fibers, and presentation of a polychromatic picture. The entire procedure was under the complete and repeatable control of the operator. Perikaryon and nuclear morphology were clearly demonstrated. The limitations of this method are that it does not provide good visualization of individual unmyelinated neuronal processes and does not permit preparation of permanent slides.     

A New Organic Dye-Based Staining for The Detection of Plant DNA in Agarose Gels

Ethidium bromide (EtBr) is used to stain DNA in agarose gel electrophoresis, but this dye is mutagenic and carcinogenic. We investigated N-719, which is a visible, reliable and organic Ruthenium-based dye, and five fluorescent alternatives for staining plant DNA. For prestaining and poststaining, N-719, GelRed, and SYBR Safe stained both DNA and PCR product bands as clearly as EtBr. SYBR Green I, methylene blue, and crystal violet were effective for poststaining only. The organic dye N-719 stained DNA bands as sensitively and as clearly as EtBr. Consequently, organic dyes can be used as alternatives to EtBr in plant biotechnology studies.     

An Indirect Radiolabelled Antibody Staining Technique for the Rapid Detection and Identification of Bacteria

Small numbers of Bacillus subtilis var. niger spores, Serratia marcescens and Francisella tularensis can be detected and identified in an hour using an indirect radiolabelled antibody staining technique with homologous rabbit antibacterial antibody and radiolabelled goat anti-rabbit globulin. Tritium is better than ¹²⁵I, ¹⁴C and ³⁵S for labelling the globulin. The reagent obtained is sensitive and adequately stable.     

The Benzidine Staining Method for Blood Vessels

Two modifications of the method are described: A. Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear. When the blood vessels appear dark blue, the specimens are fixed in acidified 70% alcohol, dehydrated, cleared and either mounted in Canada balsam as whole mounts, or embedded in paraffin, sectioned at 100 to 250µ and mounted. B. Material fixed in 10% formalin in sea-water, or in formalin hypertonic saline, is incubated at 37°C. for one hour in an aqueous mixture containing sodium nitroprusside, 0.1%; benzidine, acetic acid 0.5%, followed by a weak (0.01–0.02%) hydrogen peroxide solution for a further hour, embedded in paraffin, cut into thick sections and mounted.