1. Indications. Preservation of nuclei and other structures rich in nucleic acids, protein sulfhydryl groups, and gly-cogen.
2. Composition. 640 mL absolute ethyl alcohol, 120 mL chloroform, 40 mL glacial (99.7%) acetic acid.
Prepare fixative just before use.
3. Procedure. Slices up to 1.5 cm in thickness can be fixed. The fixation time will vary from 2-20 h. Transfer into absolute ethyl alcohol.
4. Storage. Fixed tissue should be stored in cedar oil (reagent grade or USP) or lightweight liquid petrolatum.
Formalin Solutions Formalin is a 36-40% solution of gaseous formaldehyde (HCHO) in water. One usually uses a 10% solution, which is a 4% solution of gaseous formaldehyde in water. The term "formalin" is also frequently used for the 10% solution. Thus, "formalin" and "10% formalin" have become synonymous. The 36-40% solution of formaldehyde in water is then referred to as "concentrated formalin."
In this chapter, the term "formalin" or "concentrated formalin" means a 36-40% solution of gaseous formaldehyde in water. The usual 10% formalin solution is referred to as "10% formalin solution" or "formalin solution." When tissue are referred to as "formalin-fixed" it also means that a 10% formalin solution was used.
Formalin solution is by far the most widely used fixative. For regulations designed to prevent toxic effects, see Chapter 16.
1. Indications. Ten percent formalin solution is the most widely used fixative, recommendable for most purposes; it is cheap, and requires little attention. Formalin-calcium solution is used for the preservation of phospho-lipids, and formalin ammonium bromide solution is recommended for fixation of central nervous system tissue when impregnation with gold and silver is intended. It should be noted that formalin-fixed tissues should not be frozen because during thawing, the tissue cannot absorb water normally and thus, extracellular ice crystals persist and severely interfere with subsequent microscopic study (3). Frozen section, however, are quite satisfactory.
a. Unbuffered formalin (10% solution): 100 mL formalin, 900 mL tap water.
b. Formalin-saline: 100 mL formalin, 8.5 g sodium chloride, 900 mL tap water.
Unbuffered acid formalin solutions or unbuffered neutralized formalin solutions should not be used for routine fixation and storage of tissue because of the formation of formalin pigment and its interference with various stains. Buffered neutral formalin solution is preferred. For in situ hybridization and immunohistochemistry, buffered 10% formalin solution also works quite well.
c. Buffered neutral formalin solution: A crude method is to add an excess of calcium and magnesium carbonate to unbuffered 10% formalin solution. Neutral formalin solution (buffered at pH 6.8-7.0): 6.5 g dibasic sodium phosphate (Na2HPO4), 4.0 g monobasic sodium phosphate (NaH2PO4), 10 mL formalin, 90 mL distilled water.
d. Formalin-alcohol: 100 mL formalin, 900 mL ethyl alcohol, 95%, 0.5 g calcium acetate (added if neutralization is required).
e. Formalin-calcium: 10 mL formalin, 1 g calcium chloride, anhydrous (CaCl2), distilled water to make 100 mL, piece of chalk, 3-5 cm long.
Dissolve the calcium chloride in part of the water. Add the formalin and then make to volume with water. Add the chalk to the mixture to maintain the pH, which should be approx 4.7-4.9.
f. Formalin-formic acid: 100 mL formalin, 900 mL 4 N formic acid.
g. Formalin-acetic acid-alcohol: 100 mL formalin, 50 mL glacial (99.7%) acetic acid, 850 mL ethyl alcohol, absolute.
3. Procedure. Fix slices not thicker than 6 mm in 20 volumes of formalin solution. The fixation time will be about 6-18 h. However, the tissues may remain in formalin solution for unlimited periods. Change the formalin solution until the fixative remains clear.
4. Storage. Fixed tissue should be stored in formalin solution.
h. Modified Millonig's Formalin: 100 mL concentrated formalin, 900 mL distilled water, 18.6 g monobasic sodium phosphate (NaH2PO4H2O), 4.2 g sodium hydroxide.
Procedure and storage. The solution has a pH of 7.4 and can serve as a general fixative that allows electron microscopy ofstored tissue. Sectioning ofparaffin blocks may not be as easy as after fixation with other formalin solutions.
Formalin Replacements Increasing concerns about possible toxic effects of formaldehyde gases (see Chapter 16) have created a market for commercially available solutions that closely resemble formalin but do not share many of its toxic effects. Although some seem to work well and appear to be suitable for histochemical studies (4), they generally are much more expensive and have not yet stood the test of time.
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