Cryostat sectioning

Somatostatin. Figure 1 Somatostatin-like im mu noreactivity in neurons of the periventricular hypothalamic nucleus of the rat. Coronal brain cryostat sections have been processed for im mu nohistochemistry and sequentially incubated with a primary monoclonal mouse anti human somatostatin antibody and secondary antimouse antibody conjugated with the fluorescence-dye Cy-3. Images have been taken with a Zeiss Axioplan fluorescence microscope. Scale bar, 100 pM. 

Cryostat sections should be prepared as thin as possible, 4-8 pm in thickness. Air-dry slides for 1-3 h at room temperature (see Note 7). 

Figure 13.4 Electron micrographs of the different fibres in different athletes. The fibre composition (type I and type II) of two selected top athletes, (a) A swimmer, whose speciality is the 50 metre crawl sprint, (b) A professional world-class cyclist of the roller type, (c) and (d) Cryostat sections of the swimmer s and cyclist s vastus lateralis stained for myosin ATPase, after preincubation at pH 4.3. Type I fibres stain dark, type II fibres remain unstained, (c) Almost all of the swimmer s fibres are type II. (d) Almost all of the cyclist s fibres are type I. Photographs kindly provided by Professor Hans Hoppeler, Department of Anatomy, University of Bern, Switzerland. Published in Strength and Power in Sport, ed. P.V. Komi, Blackwell Science (1992), pp.39-63.

However, connexins do not seem to be restricted to the transverse cell boundaries, since they have also been detected in several specimens at the lateral cell side. For example Oosthoek et al. [1993b] demonstrated Cx43-positive staining at the lateral cell side of human and bovine hearts (ventricles). Figure 10 shows another example from the rabbit heart, using an anti-Cx43 monoclonal antibody in cryostat sections of the rabbit left ventricle. Please note the distribution of Cx43 positivity at the transverse cell boundaries and at the lateral cell sides. 

Fig. 10. Photomicrograph demonstrating immunohistochemical staining for Cx43 in cryostat sections of the left ventricle of the rat heart. Objective x 40. neofluor achroplan, Zeiss. Magnification x 400. 

IGSS may also be performed on cryostat sections, which may then be plastic embedded for semi-thin sectioning (35)... 

Endogenous biotin, distributed in a wide variety of tissues, may also cause background staining with biotin-based immunohistochemical techniques. This biotin is especially abundant in liver, whereas it is poor in the central nervous system and adipose tissue. Endogenous biotin activity is more abundant in the cytoplasm and cryostat sections but is also present in sections of paraffin-embedded tissues. This problem is largely eliminated by using streptavidin-based methods or by sequential treatment of sections (prior to staining) with 0.01-0.1% avidin followed by 0.001-0.01% biotin for 10-20 min each. The biotin problem is discussed in more detail later in this chapter. 

Kidney tissue is fixed with paraformaldehyde-lysine-periodate by vascular perfusion (Brown et al., 1996). Tissue slices are further fixed overnight at4°C with the same fixative and stored in PBS (pH 7.4) containing 0.02% sodium azide. They are placed in 30% sucrose in PBS for at least 1 hr, and then surrounded by a drop of Tissue-Tek embedding medium on a cryostat chuck before freezing by immersion in liquid nitrogen. Cryostat sections about 5 p,m thick are cut at a chamber temperature of -25°C, collected on Fisher Superfrost Plus charged slides, and stored at —20°C until use. 

For cryostat sectioning, the tissue specimens are cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 12 hr or until they sink to the bottom of the container. They are embedded in O.C.T compound (Miles, Elkhart, IN) and frozen in N-heptane cooled to the temperature of liquid nitrogen. Alternatively, if the antigens are resistant to paraffin embedding, the specimens can be dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. 

Human skin tissue is embedded in OCT, 4-(xm-thick cryostat sections are prepared, mounted on poly-L-lysine-coated slides, and stored at -80°C. The sections are brought to room temperature (22°C), and a grease ring is drawn around the sections to limit the spread of reagents. They are rehydrated for 10 min in PBS (0.1M phosphate buffer, 0.15 M saline, pH 7.4) and treated for 15 min with blocking solution containing 2% normal swine serum and 1% BSA in PBS. 

Figure 21.2 Histochemical analysis of / -galactosidase gene expression in liver. Mice were injected with 1.6ml saline containing various amounts of pCMV-LacZ plasmid DNA. Animals were sacrificed eight hours post injection and liver sections were made using cryostat. Sections (A, B, C and D) were stained with X-gal solution followed by eosin for counter-stain. Sections (E, F, G and H) were stained by a standard hematoxylin/eosin staining method. Sections were made from animals each receiving 0 (A, E), 0.5 (B, F), 2.5 (C, G) and 25 pg (D, H) of pCMV-LacZ. (25x). (see Color Plate 14)...

Cryostat sections and cytocentrifuge preparations should be air-dried for at least 1 h but preferably overnight before immuno-staining. Before immunolabeling, cryostat sections should be fixed in cold acetone for 10 min and cytospin slides should be fixed for 90 s in a 1 1 mixture of acetone and methanol at room temperature. After fixation, follow the IGSS method for paraffin sections from step 3. Cytospin preparations are usually adequately covered by standard... 

Cryostat Sectioning of Brains Victoria ReviUa and Alison Jones... 

Most PNS antibodies can be detected using immunohistochemistry performed on cryostat sections of rat nerve tissue, usually snap frozen in iso-penthane [52, 155, 156] or fixed in formaldehyde for detection of CRMP-5 antibodies [52]. If the patient serum produces a staining pattern in nervous tissue consistent with an onconeural antibody at a dilution of 1 500 or more, this is usually regarded as positive [157] (Fig. 1). The specificity of... 

Tissue sections too thick. Cut tissue sections thinner. Formalin-fixed paraffin-embedded tissue sections should be approximately 4-6 pm cryostat section [Pg.141]

Tuson JR, Pascoe EW, Jacob D. A novel immunohistochemical technique for demonstration of specific binding of human monoclonal antibodies to human cryostat sections. J Histochem Cytochem 1990 38 923. 

Indirect immunofluorescence studies are performed on 3 11 m-tliick cryostat sections which are air dried and incubated with anti-rat intercellular adhesion molecule-1 (ICAM-1) antibody (Tamatani and Miyasaka 1990) for 60 min at room temperature. After washing the antibody binding is visualized by incubating the sections for 30 min with fluorescein isothiocyanate-labeled goat anti-mouse IgG. 

Direct immunofluorescence studies are performed on 4mm thick cryostat sections, which are incubated with fluorescein isothiocyanate-labeled goat anti-rat IgG, goat anti-rat C3, goat anti-rat fibrinogen and goat anti-rabbit IgG. (Note these procedures may also be performed on the paraffin sections using standard immunohistochemistry techniques). 

Dalton TP, Li Q, Bittel D, Liang L, Andrews GK (1996) Oxidative stress activates metal-responsive transcription factor-1 binding activity. Occupancy in vivo of metal response elements in the metallothionein-I gene promoter. J Biol Chem 271 26233-26241 Danscher G, Howell G, Perez-Clausell J, Hertel N (1985) The dithizone, Timm s sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS. A proton activation (PIXE) analysis of carbon tetrachloride extracts from rat brains and spinal cords intravitally treated with dithizone. Histochemistry 83 419 22 Danscher G, Jensen KB, Frederickson CJ, Kemp K, Andreasen A, Juhl S, Stoltenberg M, Ravid R (1997) Increased amount of zinc in the hippocampus and amygdala of Alzheimer s diseased brains a proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material. J Neurosci Methods 76 53-59... 

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