Embedding cells procedures

Conventional methods used for the preparation of biological material both for light microscopy and EM consist of fixation, dehydration, and embedding. These procedures result in increased permeability of the cell membrane with resultant loss or redistribution of mobile intracellular elements. Sections are then stained, resulting in the deposition of elements onto the specimen so that the resultant spectrum is not representative of the elemental composition in vivo (Figure 2). In consequence, such preparation methods are rarely used when the specimen is intended from the outset for microanalysis. 

The procedure for purification of Na,K-ATPase in membrane-bound form from the outer renal medulla of mammalian kidney offers the opportunity of exploring the structure of the Na,K-pump proteins in their native membrane environment. The protein remains embedded in the membrane bilayer throughout the purification procedure thus maintaining the asymmetric orientation of the protein in the baso-lateral membrane of the kidney cell in the purified preparation. This preparation has been particularly useful in studies of ultrastructure, protein conformation and for... 

The IEM double-labeling method described was performed with cells fixed in 2% paraformaldehyde and 0 04% glutaraldehyde, and embedded in Lowicryl K4M, LR White, or LR Gold. Other fixation and embedding procedures should work equally well, provided that the specimen resists the chemicals used m the silver enhancement. 

Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al 1997). 

We report a simple and direct pre-embedding technique for processing scarce biological specimens for LM and EM the samples are pre-embedded in BSA and BA, cross-linked and polymerized. The technique is compatible with a broad range of LM and EM protocols and procedures. It is applicable to tissues and cells, prepared as pellet or suspension. The technique represents an improvement over others, like the ability to visualize the samples once pre-embedded and a better resistance to histological processing. It allows a more efficient and reproducible analysis of the samples by LM and EM. The technique is... 

Histopathological analysis was performed according to a published procedure by Lipkin. The entire cecum, colon, and rectum of two animals, one each from the control group and the MCM-treated group, were removed and fixed with 10% buffered formalin (12 h), 80% ethanol (12 h), and 95% ethanol (12 h). Representative sections were taken, paraffin embedded, and 4-pm sections cut, mounted into glass slides, and stained with hematoxylin and eosin. Five slides were prepared from each tissue, each slide containing five serial sections. The number of epithelial cells per intestinal crypt over 50 intestinal crypts was counted. The number of crypts containing dysplastic epithelial cells per 50 intestinal crypts was counted. 

Fig. 3 shows SFM image of one of the sections (thickness of - 100 nm) of K562 cell embedded in epoxy resin. It should be noticed that topographical contrast and the identification of the K562 internal ultrastructure critically depend on the procedure of cell preparation before embedding (chemical fixation or high-pressure freezing and freeze-substitution). 

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