Fixation paraformaldehyde

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. 

Lanier LL, Warner NL. Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. I. Immunol. Methods. 1981 47 25-30. 

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. 

Figure 6. Cytoskeletal proteins in nucleus. HeLa cells were immunostained with anti-keratin 8 or anti-keratin 18 antibodies, (a) Paraformaldehyde-fixed cells, (b) Cells treated with detergent before the fixation. (Scale bars 10pm)...

Fixation procedures are necessary when biohazardous samples are analyzed, and are sometimes used to allow access of membrane impermeant fluorochromes, or to stabilize samples for short-term storage. Optimal fixatives are those that have low autofluorescence and do not significantly affect staining. Paraformaldehyde, at concentrations of 0.5-2%, and ethanol (70%, 4 C) are widely used fixatives for flow cytometry. Combinations of paraformaldehyde with Triton X-100 or saponin have been employed in procedures that fix and permeabilize cells. 

Formaldehyde is a colorless gas that is soluble in water (3). Commercial aqueous preparations of formalin contain 37 0% w/w solubilized gas. They also contain formic acid (<0.05%) and 10-15% methanol, which is added to prevent the polymerization of formaldehyde into paraformaldehyde (3,11). Methanol and formic acid make these solutions an unacceptable fixative for fine structures (9). Paraformaldehyde is a polymerized form of formaldehyde that dissociates at 60°C and neutral pH. Freshly prepared solutions of paraformaldehyde are preferred for most immunochemical procedures because they provide a fixative free of extraneous additives and are usually the conservative fixatives of choice when beginning the development of a fixation protocol (3,5). 

Fig. 1. (opposite page) Distribution of FITC-conjugated BSA in various fibroblast cell lines under different fixation/permeabilization regimes. (A-D) Protein distribution in living cells (A) PtKj, (B) CHO, (C) 3T3, and (D) HeLa cells. The protein is excluded from the nuclei of all cells. (E-H) Protein distribution in cells extracted for 10 min with 0.1% Triton X-100 before fixation for 30 min with 3.7% formaldehyde (E) PtKi, (F) CHO, (G) 3T3, and (H) HeLa cells. Nuclear fluorescence is seen in (E) PtKj and (G) 3T3 cells. (I-L) Protein distribution in cells extracted for 10 min with 1% Triton X-100 before fixation for 30 min with 3.7% formaldehyde (I) PtKj, (J) CHO, (K) 3T3, and (L) HeLa cells. No fluorescence is detected in the cells with the exception of some nuclear fluorescence seen in (L) HeLa cells. (M-P) Protein distribution in cells fixed for 30 min with 3.7% paraformaldehyde before permeabilization for 10 min with 0.1% Triton X-100. Fluorescence is seen primarily in the cytoplasm with the exception that nuclear fluorescence is seen in (M) PtKi and (N) CHO cells. (Q-T) Protein distributions in cells fixed for 5 min with 90% methanol, 50 vaM EGTA at -20°C (Q) PtKj, (R) CHO, (S) 3T3, and (T) HeLa cells. All cells show an overall low fluorescence, fibrous-textured cytoplasmic fluorescence, and bright staining at the periphery of the nucleus. 10 mm per scale division (black bar). (Reproduced with permission from ref. 6.)... 

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). 

Permeabilization of cells with 0.1-0.2% detergent after paraformaldehyde fixation can leave an uneven cytoplasmic distribution of the labeled proteins, and some of the larger proteins are redistributed to the nuclei. Extraction with 1 % de tergent prior to fixation removes most, but not always all of the exogenous proteins from the cell remnants. 

The use of distilled formaldehyde, not formalin, which contains alcohol, is recommended. Freshly prepared paraformaldehyde can also be used, especially if large volumes of fixative are needed for perfusion fixation. To prepare an 8% solution of paraformaldehyde, in a fume hood add 2 g of paraformaldehyde (trioxymethylene) powder to 25 mL of deionized glass-distilled water. With constant stirring, heat solution to 60-70°C. Once the solution has reached the proper temperature, continue to stir for 15 min. The solution will be milky. Add one to two drops of 1 VNaOH, with stirring, until the solution clears. A slight miUdness may persist. Cool and filter through Whatman No. 1 filter paper. This solution should be used the same day that it is prepared. 

Fixation medium Hank s BSS containing 4% paraformaldehyde plus 8-10 iM DRAQ5 . 

The IEM double-labeling method described was performed with cells fixed in 2% paraformaldehyde and 0 04% glutaraldehyde, and embedded in Lowicryl K4M, LR White, or LR Gold. Other fixation and embedding procedures should work equally well, provided that the specimen resists the chemicals used m the silver enhancement. 

A limitation is that GgnHCl tends to eliminate the antigenicity of certain intracellular structures such as microtubules. However, microtubule loss can be prevented by using low concentrations of glutaraldehyde during fixation with paraformaldehyde (Peranen et al., 1993). 

Fixation, Dehydration, and Embedding Step 1. Fixation in PFA (4% paraformaldehyde in PBS), repeat twice (cool on ice in Temperature in Microwave Oven (°C) Time (min)... 

The paraformaldehyde should be of high quality (such as that supplied by Electron Microscopy Services) and should be prepared fresh each day. Over fixation or low-grade fixatives will dramatically reduce or eliminate the antigenicity of presynaptic proteins. 

The cells or tissue sections are then fixed to preserve cellular structure and immobilize proteins in place. Fixation is often accomplished by incubating the cells/tissue in organic solvent, paraformaldehyde, or glutaraldehyde. 

Pollice, A A, McCoy, J. P, Jr., Shackney, S E, Smith, C A., Agarwal, J., Burholt, D R., Janocko, L. E, Hornicek, F J, Singh, S. G, and Hartsock, R. J. (1992) Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell-surface proteins, and intracellular proteins. Cytometry 13,432-444... 

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