Methanol/acetone fixation

Labelling of C3rtoplasmic or nuclear structures 360 Staining after glutaraldehyde fixation 361 Staining after methanol/acetone fixation 362 Staining after EGS fixation 362... 

Protocol 5.2 describes a methanol-acetone fixation technique (Pisano et al. 1993 Cenci et al. 1994) which results in very good preservation of cell morphology. Fixed cells viewed by phase-contrast optics exhibit most of the structural details that can be seen in live material. This allows analysis of unstained fixed preparations and selection of the most suitable ones for immunostaining. Remarkably, the Y loops, which are usually faint and labile in living preparations, become clearly apparent after this type of fixation. Moreover, this fixation protocol results in excellent microtubule preservation for immunostaining with antitubulin antibodies. The main disadvantage of this technique is a poor preservation of chromosome structure. In most instances, the chromosomes do not exhibit a distinct morphology and tend to coalesce into one or more masses of chromatin. 

Key words Fixation, Formaldehyde, Glutaraldehyde, Methanol, Acetone, PLP, Methacarn, Carnoy s, Bouin s... 

In this section, we will describe several procedures including fixation with paraformaldehyde, glutaraldehyde, methanol/acetone, and EGS. 

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. 

The stain/fixation method is usually used for surface markers that can withstand fixation and is followed by the application of a DNA-binding fluoro-chrome. The fixation/stain method is used not only for surface markers that can withstand fixation, but also for intracellular constituents, such as cytoplasmic proteins, nuclear membrane, and nuclear proteins. This is accomplished by using a crosslinking fixative (e.g., paraformaldehyde [PFA] or formalin) followed by a permeabilizing agent (e.g., Triton X-100, Tween-20, saponin, or lysolecithin). Some of the precipitating agents (e.g., ethanol, methanol, or acetone) can also be used for permeabilization after the initial fixation with PFA or formalin, or they can be used alone for both fixation and permeabilization (see Chapter 8). 

The preparations most often affixed to silane- (9) or poly-L-lysine-coated slides are Carnoy-fixed suspensions of metaphase cells for chromosome studies (see Chapter 47) cytospins of cultured cells followed by methanol fixation sections of formalin-fixed, paraffin-embedded tissues and frozen sections fixed with acetone or ethanol/acetic acid after cutting. Each of these approaches imparts different intracellular effects that may modify cytological detail and/or hybridization. (See Chapters 8 and 9 for a discussion of fixatives and frozen-section preparations.)... 

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... 

Frozen material or tissue-culture cells may be fixed by immersion for 10 min in 50% methanol/50% acetone at -20°C, followed by air drying. Alternatively, a 4% solution of paraformaldehyde can be prepared by dissolving 4 g of paraformaldehyde powder in 80 mL of water with gentle heating and the addition of 1 M NaOH until the powder dissolves. The solution is made up to 90 mL with distilled water, and 10 mL of a 10X PBS solution added. Fixation is for 10 min at 4°C, and slides are then rinsed in PBS. Tissues or cells which are to be used for TUNEL/ISEL should be fixed as quickly as possible, because delay causes significant artifacts. 

A high speed hybridization procedure (total, including fixation and detection, 1 h) has been described by Bourinbaiar et al. (1991). They suspended washed lymphocytes, for the detection of HIV, in a mildly hypotonic medium (1 part medium and 2 parts distilled water) and spread 25 xl ( 10 cells) in 8 mm wells of Teflon-coated multiwell glass slides and fixed the cells in Carnoy s II solution (60% methanol, 30% chloroform, 10% glacial acetic acid) for 10 min at room temperature and then for 10 min in absolute acetone. Five microliters of biotinylated probes (100 ng/ml) is added to the dried cells and the slides are covered with autoclavable plastic (cut from biohazard bag) and placed in a microwave oven. The position of the slide in the oven and the incubation time are crucial for this hybridization step. In microwave ovens with a carousel, the slide is placed about 6 cm from the center and a 150 ml beaker filled with tap water is placed in the center. The slide is then irradiated for 30 s on Defrost . The probes can be detected after a rinse with 2 x SSC. 

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