Cryosections

Leapman R D, Hunt J A, Buchanan R A and Andrews S B 1993 Measurement of iow caicium concentrations in cryosectioned ceiis by paraiiei-EELS mapping Ultramicroscopy 49 225-34... 

Nielsen, M.H., Bastholm, L., Chatterjee, S., Koga, J., and Norrild, B. (1989) Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections. Elistochemistry 92, 89-93. 

Endogenous AP activity was detected in rabbit intestine cryosections by simply adding a few drops of CL substrate [25], AP was localized in the epithelial cells... 

Figure 5 (a) Localization of endogenous AP activity in rabbit intestine cryosection by... 

Reid N, Beesley JE. Sectioning and Cryosectioning for Electron Microscopy, Elsevier, New York, 1991. 

Richter K. Aspects of cryofixation and cryosectioning for the observation of hulk biological samples in the hydrated state by cryoelectron. Scanning Microsc Suppl 1996 10 375-386. 

Erk I, Nicolas G, Caroff A, Leparet J. Electron microscopy of frozen biological objects a study using cryosectioning and cryosubstitution. J Microsc 1998 189 236-248. 

Recently, a number of groups have succeeded in cutting cryosections that were subsequently freeze dried and mounted for observation in SEM or analysis in ED AX (60) or SIMS (24,61). This technique is adequate... 

Frey B, Brunner I, Walther P, Scheidegger C, Zierold K. Element localization in ultrathin cryosections of high-pressure frozen ectomycorrhizal spruce roots. Plant Cell Environ 1997 20 929-937. 

Lazof DB, Goldsmith JKG, Rufty TW, Suggs C, Linton RW. The preparation of cryosections from plant tissue an alternative method appropriate for secondary ion mass spectrometry studies of nutrient tracers and trace metals. JMicrosc 1994 176 99-109. 

Air drying, usually with subsequent chemical post-fixation, is applicable for cell smears, cytospins and cryosections. Snap freezing (usually in liquid nitrogen) is routinely employed for tissue probes for subsequent cryosectioning. Chemical fixation is commonly carried out in aldehydes (e.g. formaldehyde) for tissue probes and cultured cell monolayers, and in acetone or alcohols (methanol or ethanol) for cryosections and cell preparations. 

This type of fixation is often used for cryosections, cell smears and cell monolyers, but can not be recommended for fixation of tissue blocks since acetone and alcohols, as opposed to aldehydes, penetrate tissue poorly. Cryosections, cell smears and cell monolyers after short (for 5 15 min) fixation in alcohols or acetone are usually air-dried (for 1 h or overnight), washed in buffered saline and directly subjected to the immunocytochemical analysis. 

In view of disadvantages associated with the use of cryosections in immunohistochemistry, the general trend is that most immunohistochemical investigations both in diagnostic pathology and in basic research studies are carried out on formaldehyde-fixed paraffin-embedded tissue sections. 

In most cases, fixation may be carried out at room temperature. Duration of formalin fixation depends on the nature and the size of the specimen, and may vary from 15 min to 24 h. Longer fixation may be associated with a partial loss of the antigenicity of the component of interest. After formalin fixation, tissue samples are washed in three changes of the buffered saline (PBS) from 15 min to 2 h, but not longer than 24 hours on the whole, since the formaldehyde fixation is partially reversible. After washing in PBS, specimens may be either snap-frozen in liquid nitrogen for subsequent cryosectioning, or dehydrated and embedded in paraffin or synthetic resin. 

For cryosectioning, tissue samples are quickly frozen with or without freezeembedding medium (e.g., Tissue Tek Miles Laboratories) and stored at 80°C until analysed. Optionally, aldehyde prefixation can also be used for tissue and organ probes before snap-freezing. Cutting of frozen tissue blocks is performed with a cryostat (a microtome mounted in a freezing cabinet). 

As for paraffin sections, it is advisable to mount cryosections also onto adhesive-coated slides in order to decrease the chances of sections dissociating from the slides in the course of immunohistochemical staining. Once mounted on slides, cryosections are air-dried and fixed, usually in acetone or methanol. Aldehyde... 

For longer storage, air-dried cryosections are best kept at 20°C or at 80°C until needed. When required, allow the slides to warm at room temperature for 5 min, then fix again in acetone (optionally) for 5 min and rinse in a washing buffer of choice. 

Fixation by rapid freezing followed by either freeze substitution or cryosectioning can also overcome some of the problems of standard immersion fixation and resin embedding. These are more specialized techniques and will not be dealt with here. Discussion of the methods can be found in Polak and Varndell (6), Hayat (7), and Verkleij and Leunissen (8). 

Slot, J. W. and Geuze, H. J. (1984) Gold markers for single and double immuno-labeling of ultrathin cryosections, in Immunolabelling for Electron Microscopy (Polak, J. M. and Vamdell, I. M., eds.), Elsevier, New York, pp. 129-142. 

As an alternative to selections on cells, other antigen sources, which better maintain the in vivo antigen expression profile and that allow appropriate in vitro selection procedures, are available. Selection on tissue cryosections may result in antibodies directed to intra- and extracellular antigens on any cell type present in the tissue section, as well as antibodies binding to matrix components [77]. 

Fig. 6 Carbohydrates in hydrogenosomes. Monocercomonas sp. after the Thiery technique (a). The hydrogenosomal membranes are positive for carbohydrates, b T. foetus cryosection labeled with gold-conjugated WGA. Hydrogenosome (H) shows that the membrane lining the peripheral vesicle, but not other portions of the organelle, is labeled. Bars = 100 nm. (Fig. 6a from Diniz and Benchimol 1998 Fig. 6b from Benchimol et al. 1996a)...

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