Fixatives methanol/acetone

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.)... 

For the phenotypic readout, 48 h after the transfection, remove media and fix cells adding 150 pi of ice-cold methanol/ acetone 10 1. 

Methanol/Acetone (1 1) stored at -20°C in a spark-proof freezer. Or 0.5% glutaraldehyde in PBS for fixing LacZ reporter cells. 

Most infected cell lines can be fixed and stained for p24 3 4 d after infection. Intracellular p24 accumulates more slowly after infection of macrophages and these cells are usually fixed 18-21 d post-infection. Cells are fixed for up to 10 min with 500 pL of cold methanol/acetone (1 1). 

Proteinase K should not be used if preparations have been fixed in methanol/acetone. 

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. 

Megestrol acetate can be recrystakhed from aqueous methanol (108). It is soluble in acetone, chloroform, and ethanol slightly soluble in ether and fixed oils and insoluble in water (107). Additional spectral and physical data have been pubHshed (62). 

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... 

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). 

Overfixation of specimens is also recognized by difficulty in sectioning because of excessive hardness of the tissue. This problem arises when tissues are fixed with formulations containing ethanol, methanol, or acetone. Excessive dehydration with an organic solvent may also cause tissue hardness, especially of small specimens (1-2 mm). Such... 

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... 

Compositional mixture experiments involve some specialist techniques and a whole range of considerations must be made before designing and analysing such experiments. The principal consideration is that die value of each factor is constrained. Take, for example, a du-ee component mixture of acetone, methanol and water, which may be solvents used as the mobile phase for a chromatographic separation. If we know diat diere is 80 % water in the mixture, there can be no more than 20 % acetone or methanol in die mixture. If there is also 15 % acetone, die amount of methanol is fixed at 5 %. In fact, aldiough there are three components in die mixtures, these translate into two independent factors. 

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