A methacrylate embedding procedure developed for immunolocalization on plant tissues is also compatible with in situ hybridization.

A recently described method that uses methacrylate embedding of aldehyde fixed plant tissues allows the immunolabelling of a range of antigens (Baskin et al. 1992). We have tested whether the same embedding procedure is also compatible with in situ hybridization. For this purpose we have used 2- 5 μm sections of methacrylate embedded plantlets of Arabidopsis thaliana. After removal of the resin the sections were prepared for in situ hybridization following standard procedures. Three different digoxygenin (dig)-labelled probes were used, recognizing RNAs coding for the chlorophyll a/b binding protein cab-140, the β-tubulin tub5 and meri a member of the meri-5 family. Each of the probes shows the labelling pattern expected from the literature. Moreover, the method allows a good structural preservation of very fragile tissues, in contrast to paraffin embedding. We conclude that methacrylate embedding, allowing both immunolabelling and in situ hybridization with high resolution and structural preservation, offers a high potential for the functional analysis of genes and proteins in plant development. This is especially true for Arabidopsis thaliana, a widely used model species where it seems to be the method of choice.     

A modified staining technique for arbuscular mycorrhiza compatible with molecular probes

The effects of the different steps of the root staining on the arbuscular mycorrhizal (AM) fungal rDNA extraction and amplification have been assessed. The results obtained using molecular techniques are compared with those obtained from fresh, non-stained leek roots. A modified staining procedure that eliminates heating, the use of hydrochloric acid and trypan blue, has been proved to be the most adequate to observe the AM colonisation in different plant species with/without lignified roots allowing at the same time the subsequent rDNA extraction and amplification from the stained roots. The staining technique decreased the sensitivity of the process and a higher number of roots had to be used to obtain enough material for a positive amplification. The extraction and amplification process was reliable up to 3 days after staining. A week after staining, the amplification was not dependable and after 2 weeks there was no amplification from stained material.     

Use of hydrogen peroxide-egg albumin to eliminate nonspecific staining in immunoperoxidase techniques.

The application of hydrogen peroxide and egg albumin to paraffin sections before immunostaining prevents nonspecific staining by immunoperoxidase techniques. This method is more effective than pretreating secions with normal sera, or using either egg albumin or hydrogen peroxide separately, or using diluted antisera with prolonged incuabations in the staining procedure.     

A modified Coomassie Brilliant Blue staining method at nanogram sensitivity compatible with proteomic analysis

A more sensitive and convenient Coomassie Brilliant Blue (CBB) staining method for visualizing proteins was developed. Compared with the modifications include the supplement of 10% (v/v) methanol into the fixing solution, an increase of an additional sensitization step and CBB raised from 0.1 to 0.125%. The improved method can detect proteins at nanogram level. The improved method is more sensitive than Blue Silver and more convenient than the Silver protocol. Mass spectrometry results confirmed that it is suitable for subsequent proteomic research.     

A modified mallory-cason staining procedure for large cryosections.

The classic Mallory-Cason staining procedure has been modified for application to sections "on tape" obtained from large deep frozen tissue specimens. These 20 microns cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.     

A hematoxylin staining procedure for maize pollen grain chromosomes

Studies of postmeiotic chromosome behavior have been impeded by the thick exine and abundant starch grains of maize pollen. Staining pollen grain chromosomes with acetocarmine is tedious and gives inconsistent, often unsatisfactory results. A hematoxylin stain, used in conjunction with the clearing agent chloral hydrate, has been successfully used by the authors to stain chromosomes, nuclei and sperm cells of the maize pollen grain. An ethanol-formaldehyde fixing fluid is used to fix and preserve the pollen samples. The procedure, which is rapid and simple, gives excellent preparations with both fresh and fixed material. Stained preparations do not get darker with time, as is typical of other hematoxylin stained materials.     

A stable propidium iodide staining procedure for flow cytometry

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.     

Standardization of the Romanowsky staining procedure: an overview

Commerically available Romanowsky blood stains are variable mixtures of thiazein dyes and brominated fluorescein derivatives with varying degrees of metallic salt contamination in a number of different solvent systems. There is a need for standardized Romanowsky stains of constant composition, which, when used in conjunction with a carefully controlled specimen preparation technique, should give consistent performance. Such a preparation system would be of great value to hematologists in general and would be essential to the validity of data obtained by the digital processing of blood cell images. It is possible to prepare standardized Romanowsky stains as mixtures of two or three dye components, namely, eosin Y, azure B and methylene blue, although azure B has only recently become commercially available at an acceptable degree of purity. The logistic problems of stain standardization are discussed.     

Influence of embedding media in prolactin labelling with immunogold techniques

Summary Immunogold labelling of prolactin in three different embedding media was compared. The polymeric prolactin in secretory granules was labelled in the three media, however, acrylic monomers (Lowicryl K4M and LR White) provided a more intense labelling with higher dilutions of the primary antibody, compared to the labelling in the epoxy resin (araldite). An intense labelling of monomeric prolactin in Golgi complex was detected only in acrylic embedments, and the labelling on the rough endoplasmic reticulum was significant only in LR White embedded tissues.     

A rapid whole-mount staining procedure for nuclei of mammalian embryos

A rapid staining procedure for examining nuclei of mammalian embryos is described. Embryos are placed on a glass slide, counterstained with trypan blue, stained with Hoechst 33342, and embedded between the slide and coverslip in Permount. The nuclei of the embryos fluoresce brightly when examined by fluorescence microscopy immediately after staining or after extended storage. The technique has proved to be an effective tool for studying the development of cow, hamster, mouse, pig, rabbit, and sheep embryos.