Tissue preparation methods

Larsson L. Tissue preparation methods for light microscopic immunohistochemistry. Appl lmmunohistochem 1993 1 2-16. 

Brandtzaeg P (1982) Tissue preparation methods for immunocytochemistry. In Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry, vol. 1. Academic, New York, pp 1-76... 

The protocols described below illustrate (1) frozen section sample preparation, (2) hematoxylin and eosin (H E) tissue staining, and (3) automated LCM. Alternative tissue preparation methods, such as ethanol or formalin fixation with paraffin embedding, are acceptable for RNA and DNA analysis... 

Edwards, F. A. and Konnerth, A. (1992) Patch-clamping cells in sliced tissue preparations. Methods Enzymol. 207, 208-222. 

This chapter contains two main parts, the first part (section 5.2) deals with sample preparation for tissues, and the second part (section 5.3) deals with sample preparation for cells. In section 5.2, tissue preparation methods for spectroscopic analysis such as cryopreservation (section 5.2.2), fixation (section 5.2.3) and embedding (section 5.2.4) are described and a number of studies investigating the... 

In order to reduce or eliminate off-line sample preparation, multidimensional chromatographic techniques have been employed in these difficult analyses. LC-GC has been employed in numerous applications that involve the analysis of poisonous compounds or metabolites from biological matrices such as fats and tissues, while GC-GC has been employed for complex samples, such as arson propellants and for samples in which special selectivity, such as chiral recognition, is required. Other techniques include on-line sample preparation methods, such as supercritical fluid extraction (SFE)-GC and LC-GC-GC. In many of these applications, the chromatographic method is coupled to mass spectrometry or another spectrometiic detector for final confirmation of the analyte identity, as required by many courts of law. 

Analysis of samples. For STEM analysis all samples were prepared using the tissue grinding method with suspension of the fine... 

Displacement binding experiments with 3H-PCP were conducted with minor modifications of the filtration method described in the literature (Zukin and Zukin 1979 Vincent et al. 1979 Hampton et al. 1982) in crude tissue preparations of rat whole brain homogenates in 5 mM Tris-HCl (pH 7.4). 

As a gold standard, fresh tissue prepared by snap-frozen method, cut by cryostat, and fixed in acetone, ethanol, or other non-cross-linking fixatives, has been generally accepted as reliable. 

Leong3 postulated that internal controls were required to optimize the variable influences resulting from aspects of tissue preparation and factors intrinsic to the staining method. Various controls have been employed, includ-... 

The last sample preparation method for IMS is the transfer of a tissue section onto the PVDF membrane. Proteins in the section can be transferred onto the PVDF membrane and then analyzed on the membrane. The advantage of this method is that the enzyme can be digested for MS" measurement, because the information on protein localization in the organization is fixed on the membrane.5,20 This technique can denature, reduce, and digest the proteins in the tissue section efficiently and remove the salt from the tissue. This increases the efficiency with which biological molecules are ionized, making it possible to obtain sensitive mass imaging spectra. 

Immunocytochemical methods have been widely applied to visualize proteins, carbohydrates, or lipids in sectioned material. The advantage of using immunocytochemistry is to be able to localize the molecules of interest within the tissue. Several procedures have been described. Basically, these procedures can be split into four main steps that are described in subheadings (1) tissue preparation, (2) the primary antibodies, (3) the visualization of the target, and (4) enhancement of signals with antibody complexes. In addition, a protocol for alkaline phosphatase will be presented in detail in Subheading 5. The terms primary and secondary antibodies refer to the order in which they are applied to the target. The immunocytochemical procedures are not limited to sectioned... 

This technique is simple in basic principle. Material is first rapidly frozen to the temperature of liquid nitrogen. It is then fractured, cryo-planed to produce a flat surface for analysis, and transferred to the cryo-stage of an SEM. It is analyzed while still frozen, and thus ion movement during tissue preparation should be minimal. A more detailed scheme of a typical procedure (45,46) is given in Subheading 3.4.2.1. This is undoubtedly the most popular microanalytical method with plant scientists at present, and as Table 1 shows it has been applied to a wide range of tissues and research topics (46-53). Recent developments include a... 

In order to achieve the firm fixation of the artificial cornea to host tissues, composites of collagen-immobilized poly(vinyl alcohol) hydrogel with hydroxyapatite were synthesized by a hydroxyapatite particles kneading method. The preparation method, characterization, and the results of corneal cell adhesion and proliferation on the composite material were studied. PVA-COL-HAp composites were successfully synthesized. A micro-porous structure of the PVA-COL-HAp could be introduced by hydrochloric acid treatment and the porosity could be controlled by the pH of the hydrochloric acid solution, the treatment time, and the crystallinity of the HAp particles. Chick embryonic keratocyto-like cells were well attached and proliferated on the PVA-COL-HAp composites. This material showed potential for keratoprosthesis application. Further study such as a long-term animal study is now required [241]. 

To obtain tissue preparations whose constituents were maintained as closely as possible to their state in vivo, the material had to be fixed, i.e. the enzymes inactivated so that cell structures were instantaneously preserved, an almost unattainable ideal. Formalin was the favored fixative, but others (e.g. picric acid), were also employed. Different methods of fixation caused sections to have different appearances. Further artifacts were introduced because of the need to dehydrate the preparations so that they could be stained by dyes, many of which were lipid-soluble organic molecules. Paraffin wax was used to impregnate the fixed, dehydrated material. The block of tissue was then sectioned, originally by hand with a cut-throat razor, and later by a mechanical microtome. The sections were stained and mounted in balsam for examination. Hematoxylin (basophilic) and eosin (acidophilic) (H and E staining) were the commonest stains, giving blue nuclei and pink cytoplasm. Eosinophils in the blood were recognized in this way. 

---