Glutaraldehyde fixation

The effect of double fixation on the extraction of lipids during dehydration and infiltration was studied by Carlier (1968) in rat intestine. When partial dehydration was used according to Idelman (1964, 1965), double fixation (glutaraldehyde followed by osmium tetroxide) reduced the loss of label of 26.6%, encountered with tissues fixed in osmium tetroxide alone, to 12.2%. This improvement was not noted when the tissues were subjected to complete dehydration, which included 100% ethanol and propylene oxide. Another point evaluated in that study was the effect of temperature on the procedure of preparation. It became apparent that when isolated labeled chylomicrons are subjected to partial dehydration the loss of label into the infiltrating resin is reduced from 20.5% at 25° to 11.5% at 4°. Relatively low extraction of lipids from rat intestine labeled with linoleic acid was reported by Buschmann and Taylor (1968) during dehydration in graded ethanols at 0°, and considerably more label was lost to propylene oxide and propylene oxide Araldite mixture, as the last steps were carried out at room temperature. Another study dealing with retention of lipids by the intestine (Dermer, 1968) showed that neutral lipids and phospholipids were extracted to the same extent, and the total loss amounted to about 16%. 

Fig. 1. A section through a chloroplast of a leaf of a maize plant grown in darkness and then exposed to light for 16 hours. Fixation glutaraldehyde-osmium. Post-stain uranyl acetate. Ribosomes (R) are seen in and outside of chloroplast s small densely stained particles. G, one of the many grana.

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. 

Hassell, J., and Hand, A. (1974) Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of cross-linking and ultrastructure obtained with dimethylsuberimidate and glutaraldehyde./. Histochem. Cytochem. 22, 223-239. 

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. 

Fixation was carried out at room temperature or at 4°C for 1-2 h in 0.1 Mcacodylate buffer (pH 7.4) containing 1% glutaraldehyde and 1 % paraformaldehyde. The pH of the ZIO mixture was approximately 5.7. For comparison, some tissues were impregnated in solution, the pH of which had been raised to 7.0 with NaOH. 

Pursel, V. G. and Johnson, L. A. (1974). Glutaraldehyde fixation of boar spermatozoa for acrosome evaluation. 

Autofluorescence of cells often complicates the studies with fluorescence microscopy (especially the application of green fluorescent substances). There are different reasons for the occurrence of this phenomenon (157) (i) the fluorescent pigment lipofuscin, which settles with rising age in the cytoplasm of cells (ii) cell culture medium, which often contains phenol red that increases autofluorescence (iii) endogen substances such as flavin coenzymes [flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN) absorp-tion/emission 450/515nm], pyridine nucleotides [reduced nicotinamide adenine dinucleotide (NADH) absorption/emission 340/460nm] or porphyrine (iv) substances taken up by cells (as mentioned above filipin) and (v) preparation of the cells fixation with glutaraldehyde increases autofluorescence. 

Goldberg M and Septier D (1985) Improved lipid preservation by malachite green-glutaraldehyde fixation in rat incisor predentine and dentine. Arch Oral Biol 30, 717-726. 

Fixation in glutaraldehyde prodnces better morphology, but induces a great deal of autofluorescence, and limits cytoplasmic and nnclear permeability. A protocol utilizing glutaraldehyde followed by borohydride treatment has been previously described that is applicable also for electron microscopy of cultnred cells (4). Some areas of the cell are relatively impermeable with this approach, bnt this is an excellent choice of fixative protocol for microtnbnle morphology. 

Jamur, M. C., Faraco, C. D., Lunardi, L. O., Siraganian, R. P., and Oliver, C. (1995) Microwave fixation improves antigenicity of glutaraldehyde-sensitive antigens while preserving ultrastructural detail. J. Histochem. Cytochem. 43, 307-311. 

Colors and intensity of bands may differ after formaldehyde fixation compared with those obtained by glutaraldehyde fixation. Formaldehyde yields blackish and glutaraldehyde gives brownish bands. 

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