Specimen preparation fixation

III. Transmission electron microscopy of radish seeds Transmission electron microscopy (TEM) of radish seeds was done as listed below For TEM preparations, the specimens after fixation and dehydration, were embedded in Epon 812 resin (Luft, 1961). Thick sections (ca. 1mm each) were stained with 0.1% toluidine blue and observed with a Zeiss light photomicroscope. Thin sections, obtained with a diamond knife on a Supernova microtome, were sequentially stained at room temperature with 2% uranyle acetate (aqueous) for 5 min and by lead citrate for 10 min (Reynolds, 1963). Ultrastructural studies were made using a Philips CM12 transmission electrone microscope (TEM) operated at 80 KV. 

Finally, the localizations of low-molecular-weight compounds requires special specimen preparation techniques, as these compounds are often diffusible, water- or organic-solvent soluble, and solubilized by conventional fixation and dehydration procedures. The reader is referred to ref. (12) for the processing of cells and tissues for the cytochemical and histochemical localization of these compounds. 

Sources of specimens, the type of preparation, and the fixation needed for immunocytochem-istry. Almost any type of specimen preparation or fixative can be used. The gentlest fixation is air-dried-acetone, which consists of first air-drying the slide followed by a brief acetone postfixation Chapters 9-12). 

Biological autofluorescence in mammalian cells due to flavin coenzymes (FAD and FMN absorption, 450 nm emission, 515 nm) and reduced pyridine nucleotides (NADH absorption, 340 nm emission, 460 nm) can be problematic in the detection of fluorescence probes in tissues and cells. Fixation with aldehydes, particularly glutaraldehyde, can result in high levels of autofluorescence. This can be minimized in fixed cells by washing with 0.1% sodium borohydride in phosphate-buffered saline (5) prior to antibody incubation. Problems due to autofluorescence can be minimized by selecting probes and optical filters that maximize the fluorescence signal relative to the autofluorescence. Other factors that limit IF include the performance of the detection instrument (i.e. how well the microscope has been calibrated and set), the specificity of the antibodies, and the specimen preparation. 

Interfacing Light Microscopy with TEM Fixation and Specimen Preparation for TEM Selective Staining of Nuclear Components Image Analysis and Enhancement Three-Dimensional Reconstructions by TEM... 

Figure 7 Preparation of Cryo-TEM specimen (a) fixation of the grid using a pair of tweezers, (b) removal of excel solvent, (c) close-up of the specimen grid, and (d) freezing of the specimen in a coolant.

The major structural unit of interest in emulsions, microemulsions, colloids and latexes is the particle. It is well known that the particle shape, size and distribution of a latex controls the properties and end use applications. Many latexes are manufactured with a controlled and sometimes monodisperse distribution of particle sizes. Polymer liquids, in the form of emulsions and adhesives, are wet and sticky, and therefore specimen preparation for electron microscopy is very difficxilt. As a result of the importance of the determination of particle size distribution, microscopy techniques have focused on specimen preparations which do not alter this distribution or which alter it as little as possible. Methods have included special cryotechniques (Section 4.9), staining-fixation methods (Section 4.4), microtomy (Section 4.3) and some simple methods (Section 4.1) such as dropping a solution onto a specimen holder. This section is meant to provide a brief survey of the types of microscopy applications which have been foimd useful in the evaluation of emulsions and latexes. 

TTHIS CHAPTER IS FOR INDIVIDUALS who have not done electron microscopy (EM) before, but it also contains some new information about specimen preparation that can benefit experienced electron microscopists. It covers the materials and methods necessary to do routine thin sections of embedded specimens however, it does not cover specialized EM techniques such as freeze-fracture, rotary shadowing, negative staining, scanning EM, and serial sectioning. Likewise, this chapter focuses mainly on embryos because a description of the fixation methods for all the various types of fly tissues would require an entire book. This chapter is divided into three main sections ... 

The fixative system generally used is a two-vial technique with one vial containing 5 to 10% buffered Formalin and the other vial containing polyvinyl alcohol (PVA) fixative. A portion of the specimen is added to the fixative in a ratio of approximately 3 parts fixative to 1 part specimen and thoroughly mixed to ensure adequate fixation. An alternative to Formalin is Merthiolate-iodine-formaldehyde (MIF), which fixes and stains at the same time. If unfixed specimens are processed in the laboratory, fecal films may be prepared and immediately fixed in Schaudinn fixative. 

It is that simple. Understanding penetration is vital to seeing why the preparation of specimens sometimes (often) turns out other than what we expect or desire. Penetration of all other fluids after the fixative also must be considered. One of the purported consequences of fixation is to make the tissue more... 

Body fluid specimens will be prepared and stained and the morphologic characteristic of the cells and the environment in which these cells are found will be examined by light microscopy. To achieve this, a representative cell sample must be obtained and adequate cell fixation is a prerequisite. Proper identification of the specimen and protection of the specimen s integrity are essential. Finally, pertinent patient clinical history is important for accurate specimen interpretation. 

In order to evaluate binding, uptake, and intracellular localization of targeted drugs via CLSM, nonfluorescent molecules of interest, as well as the appropriate structures of the specimen, have to be labeled with suitable fluorophores. This technique, however, requires a number of preparative steps, including fixation and permeabilization of the sample, staining and counterstaining, as... 

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