Fixation ethanol

This protocol describes one of several useful procedures for constructing DNA content frequency histograms that yield information about cell-cycle phase distributions. The method uses ethanol-fixation to prepare cells for staining with PI. 

Battifora, H. and Kopinski, M. (1986) The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation. J. Histochem. Cytochem. 34,1095-1100. 

To distinguish diffusible from bound antigens, cryostat sections are often preferred. Pre-fixation diffusion artefacts (e.g., passive uptake of non-specific antigens by cells, particularly of the lymphoid system Mason et al., 1980), which occur quite often, may be revealed by double EIH staining (e.g., k and X chains should normally not be present in the same cell) and counteracted by rapid fixation or removal of diffusible antigens by washing with cold saline. Postfixation diffusion artefacts are also frequently noticeable, e.g. after ethanol fixation (Brandtzaeg, 1982). 

This first study encouraged us to continue to obtain additional lysin sequences. We found that ethanol fixation (50%-90%) of fragments of abalone testis preserved the lysin mRNA, making possible procurement of testis samples from approximately 30 species world wide. Following the synthesis of universal lysin primers in the 3 and 5 untranslated regions of lysin cDNA, it was a simple process to obtain lysin sequences from an additional 25 species using RT PCR (Lee et al., 1995 Lee and Vacquier, 1995). 

A comparison of the relative content of the GAPDH template in the samples obtained from each fixation condition is shown in Fig. 4. The highest yield of GAPDH mRNA was obtained with 70% ethanol fixation. By comparison, 100% methanol fixation produced 40% less yield of GAPDH mRNA. The 100% ethanol and 70% methanol fixations yielded more GAPDG mRNA than from 100% methanol but less than with 70% ethanol. 

Soukup J, Krskova L, Hilska I, Kodet R (2003) Ethanol fixation of lymphoma samples as an alternative approach for preservation of the nucleic acids. Neoplasma 50 300-304... 

Hu SP, Yang JS, Wu MY, Shen ZY, Zhang KH, Liu JW, Guan B (2005) Effect of one-step 100% ethanol fixation and modified manual... 

The use of this wax for immunohistochemistry has previously been reported in combination with both formalin fixation (4) and acid-ethanol fixation (5). Its routine use for immunohistochemistry was described in 1991 (6), and it has been used in my laboratory as the method of preference for immunohistochemistry for 11 yr (e.g., 7,8). It can be used for any immunostaining procedure, e.g., immunofluorescence or immunoperoxidase, with or without counterstaining, or combined with In situ hybridization (ISH) (9). 

Roholl, P. J. M., Dullens, H. F. J., Kleijne, J. Dubbink, E. J., and Den Otter, W. (1991) Acid ethanol fixation and polyester waxembedding combines preservation of antigenic determinants with good morphology and enables simultaneous bromo-deoxyuridine (BRDU) labeling. Biotech. Histochem. 1, 55-62. 

Pleshko et al have shown that 70% ethanol fixation of 35-day-old embryonic rat femur gave rise to FTIR spectra that exhibited Amide I and II peaks that were shifted to lower frequencies (1647cm and 1546cm respectively) compared with unfixed rat femur, which exhibited Amide I and II peaks at 1651 cm and 1550cm It was concluded that evaluation of protein structure in this tissue should be limited to snap-frozen samples, or formalin-fixed tissues where there were no observable shifts in the Amide I and II peaks. Although this does not conform to the observations made by Faolain et al., this difference may be due to the different types of tissue used in each study, calcified tissue in Pleshko et al and non-calcified tissue in Faolain et al ... 

Table 12.19 compares free run and press juice composition for traditional (crushed grapes) and carbonic maceration winemaking. In carbonic maceration press wines, the alcohol content is higher (caused by ethanol fixation) and the acidity lower (due to malic acid degradation). These wines also have lower concentration of phenolic componnds and other extracted components their dissolntion is diminished. 

HL-60 cells were treated with indicated concentimtions of camosic acid, camosol or ursolic acid for 18 h (right column) and treated with camosic acid (30 fiM), camosol (20 tiM) or ursolic acid (40 tiM) for different time periods (left column). After 70% ethanol fixation and propidium iodide (PI) staning, DNA content was analyzed by FACs laser flow cytometer. Percentages represent apoptotic ceils among each group. Data represents mean SE of triplicate tests. 

Ciancio, G. Pollack, A. Taupier, M. A. Block, N. L. Irvin, G. L., HI, Measurement of cell-cycle phase-specific cell death using Hoechst 33342 and propidium iodide preservation by ethanol fixation. J. Histochem. Cytochem. 1988, 36, 1147-1152. 

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. 

Figure 12.3 Hydroxymethyl adducts from formaldehyde fixation form highly reactive imines in the presence of ethanol during tissue processing, giving off a molecule of water (upper). While still in alcohol, imines may either form more complex ethoxymethyl adducts or will cross-link to neighboring reactive groups (lower).

EFFECT OF FIXATION AND ETHANOL DEHYDRATION ON PROTEIN STRUCTURE... 

Fowler CB, O Leary TJ, Mason JT. Modeling formalin fixation and histological processing with bovine ribonuclease A effects of ethanol dehydration on reversal of formaldehyde-induced cross-links. Lab. Invest. 2008 88 785-791. 

Chemical fixation for transmission electron microscopy prepares cells for the preservation of damage due to subsequent washing with aqueous solvents, dehydration with organic solvents such as ethanol or acetone, embedding in plastic resins, polymerization of the resins by heat, exothermic catalysts, or ultraviolet radiation, and imaging with high-energy electron beams in an electron microscope. 

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