Fixatives glutaraldehyde

Instead of whole-mount cells, it is also possible to prepare thin sections. Cells are grown on plastic coverslips, fixed (glutaraldehyde concentration lowered to 2%), hybridized and the label detected as described above. The cells are then postfixed with 2% OSO4 in PBS for 30 min and dehydrated in graded alcohol. They are then passed through 100% alcohol/Epon (1 1) for 1 h at room temperature and 1 h in 100% Epon. Beem capsules filled with Epon are quickly inverted onto the surface of the coverslip and oven-baked at 60°C for 48 h. The Beem capsule and coverslip are removed followed by routine ultrathin sectioning and processing. 

Fig. 18. Combined experimental cytology and electron microscopy of the same bivalent in a grasshopper Melanoplus differentialis) spermatocyte. A, The cell is shown in life at the lower left (O.O-minute print). The kinetochores of a bivalent which had been detached from the spindle by micromanipulation and moved to the cytoplasm are designated by arrows labeled and "2 1.2 minutes later the cell was fixed (glutaraldehyde post-fixed in OsO ) and is shown after embedding in Epon (1.2-minute print). Careful analysis shows that the half-bivalent labeled had moved counter-clockwise 1 to 2 ju by the time of fixation the second half-bivalent had not moved. The bulk of the figure shows a survey electron micrograph of the same cell with the kinetochoric ends of the manipulated bivalent identified by arrows and showing two other bivalents also visible in the light microscopic prints. Light microscopic prints, XI,000 electron micrograph, X7,500.

For an excellent review of tissue and section preparation methods in plants, see ref. 31. Fixation conditions will vary with the tissue, the cell-type and its permeability to the fixative. Glutaraldehyde does not easily penetrate leaf cuticle, but is immediately available to stem cross-sections. Fixation with a 2.5% glutaraldehyde in 0.1 M Na2HP04 pH 7.0 for 2 to 3 minutes on ice seems to leave a reasonable amount of GUS activity, when followed by extensive washing. Fixation should be tested empirically in any new system. Formaldehyde seems to be a more gentle fixative than glutaraldehyde, and can be used for longer times. We have successfully used 0.3% formaldehyde in 0.3 M mannitol, 10 mM MES, pH 5.6 for 30 to 45 minutes at room temperature for protoplasts. This should be followed by several washes, usually in phosphate buffer or osmoticum. 

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

Fixation tissue samples for immuno histochemistry were fixed in 2% paraformaldehyde, 0.25% glutaraldehyde and 3% sucrose buffered with 0.05M phosphate buffer pH 7. After incubation for 2 hours at 25 °C and 63 hours at 5°C the specimens were washed 3 x 20 min. in phosphate buffer pH 7. Dehydration was carried out using series of ethanol washings 50, 70, 80, 96% followed by 3 x in 99% (V2 hr in each). After additional treatment with 2x2 hrs in petroleum ether (shellsol D70k, Q7712) and 2 x 2 hrs in paraffin with 7% beeswax, the samples were embedded in paraffin. Cross sections of 12.5 /im were made on a Supercut 2050 Reichart Jung pyramitome. 

II. Scanning electron microscopy of radish seeds For scanning electron microscopy (SEM) observations, seed coats and endosperms were fixed in 3% glutaraldehyde in 0.065 M phosphate buffer (pH 7.4) for 2 h at... 

Catalase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated Teflon membrane served as enzyme electrode to determine hydrogen peroxide [248], The electrode response reached a maximum when 50mM phosphate buffer was used at pH 7.0 and at 35°C. Catalase enzyme electrode response depends linearly on hydrogen peroxide concentration between 1.0 X 10-5 and 3.0 X 10-3 M with response time 30 s. 

Formaldehyde or glutaraldehyde Labeled mannose Polysaccharide Considerable 20% loss of radioactivity with either fixative Vanha-Pertulla and Grimley (22)... 

Harvested samples should be immediately prepared for the SEM by fixation in a 0.5% osmium tetroxide solution in the dark for 1 h. If the sample is collected in the field, store in buffered 3% glutaraldehyde solution (pH 7.2) until samples can be fixed. 

Diatom colonies formed in polystyrene culture dishes were fixed by adding a 2.5% glutaraldehyde solution to culture dish. 

Note We recommend that after performing step 1 above that the specimens be rinsed three times in a phosphate buffer (pH 7.2), which will remove any residual glutaraldehyde before beginning the dehydration series. Garduno etal. (13) also use a second technique in which they cut small pieces of medium containing diatoms and immediately fix the pieces in liquid nitrogen. The pieces are freeze-dried and then gold sputter coated. 

Fig. 4. Comparison of osmium-fixed (A) and glutaraldehyde-prefixed and osmium-postfixed (B) pollen tubes, (from Dashek and Rosen [5H], with permission.) (A) = 5800X (B) = 9300x.

The tissues were fixed in 0.05 M cacodylate buffer containing 2.5% glutaraldehyde and 1.5% formaldehyde (pH 7.0) for 16 h. The ZIO mixture was prepared as follows 3 g zinc (powder) and 1 g resublimed iodine crystals were dissolved in 20 mL distilled water. After stirring for 5 min, the zinc was filtered off. The filtered solution was mixed with an equal volume of 2% 0s04 solution and the solution used immediately. Treatment with the ZIO mixture was carried out for 4 h at room temperature. 

Animals were fixed by perfusion with 0.1 M phosphate buffer containing 2% glutaraldehyde and 4% saccharose (pH 7.4). Treatment with the ZIO solution was carried out for 20 h at 4°C. 

Epon 812 (Polybed) Formvar Glutaraldehyde Ilford L-4 emulsion with appropriate safelight Osmium tetroxide Phosphate Dibasic Monobasic Propylene oxide Sodium thiosulfate fixing solution... 

Fix pollen tubes 30 min in 6.25% glutaraldehyde in phosphate buffer or veronal-acetate buffer, pH 7.4. 

Fix with 4% glutaraldehyde buffered with 0.1 MNa-cacodylate, pH 7.2 for 24 h in a refrigerator (the solution in this case also contained the appropriate concentration of divalent cations). 

Figure 2. Scanning electron micrograph of a mesophyll cell of a dormant cotyledon of Buffalo gourd (Cucurbita foetidissima). Tissue was fixed in aqueous glutaraldehyde, dehydrated with ethanol and critically point dried. Note cell wall (W) and intracellular components including protein bodies (P) and emptied spherosomes that appear as a cytoplasmic reticulum.

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