With a few exceptions (such as enzyme activity in tissue cultures, frozen or frozen-dried preparations), histochemical investigations on living cells are not performed because it is difficult to maintain the tissue and/or cell integrity under such conditions.
As a result, it must be ‘fixed’ or preserved in such a way that subsequent examinations can be made to determine its micro-anatomy and allow localization of its various chemical constituents. Unfortunately, as will be seen below, it has not been demonstrated that all of these functions can be accomplished with a single fixing reagent or group of reagents. As a result, the problem of selecting a fixative that will preserve
- The gross and microscopic anatomy of the tissue to be examined
- The chemical compounds to be investigated, particularly their histochemically reactive groups, and, if possible
- Other classes of compounds that may become important during the course of an investigation
Adequate and complete fixation is the foundation of all good histological preparations. Faults in fixation cannot be corrected later, and the finished section is only as good as its primary fixation.
Tissues must be fixed as soon as possible after death or removal from the body, which is why screw-capped specimen jars containing appropriate fixatives should be kept permanently wherever tissues for histological examination are taken on a regular basis, such as in the operating room, post-mortem room, or animal house. The fluid in the jars should be 15-20 times the bulk of the tissue to be fixed. The easy availability of such jars will eliminate the need for tissues to dry. To avoid over-fixation in intolerant fixatives, specimens should be sent to the histology laboratory as soon as possible.
Tissues chosen for sectioning should be thin enough to be adequately fixed throughout in a reasonable amount of time. The volume of fixative required is determined by the overall bulk of the tissue; the speed of fixation is determined by the thickness. The type of microtome used will often determine the length and breadth of the tissue taken; the best thickness for routine use is 3-5 mm.
In addition to immersion, injection of an appropriate fixative, usually formol saline, is generally recommended for complete fixation of whole organs. Only this combination of perfusion and immersion can adequately repair the brain.
Aims and effects of fixation
Fresh tissue left in a warm environment (at room temperature) will liquefy and emit a foul odor due to putrefaction and autolysis. To prevent such deterioration, the examination of fresh tissue necessitates the use of a preservative. However, simply preserving tissue is insufficient; this can be accomplished by quickly freezing at low temperatures, but such tissues must be kept frozen in airtight containers; thawing will restart the deterioration process.
Fixation must preserve the tissue in such a way that it retains as much of its original form as possible while still allowing for certain investigative procedures. Early researchers in this field demanded that the tissue be sufficiently hard to allow for the cutting of thin slices by hand.
Although modern embedding techniques provide adequate support for tissue and allow for the cutting of thin slices (or sections), the hardening effect of fixatives is useful. We frequently use tests that would destroy a living cell, but fixed tissue sections can withstand such treatment with ease. Many of the apparent by-products of fixation will be shown to be useful in subsequent investigations.
The goal of fixation is to preserve tissue in the most lifelike manner possible. This preservative must also be able to withstand a variety of techniques without losing its integrity. The most common side effects are listed below.
Inhibition of Autolysis and Putrefaction
Autolysis and putrefaction are two of the most common causes of poor cellular detail in histological preparations.
Cell lysis or dissolution caused by enzymatic action, most likely as a result of lysosome rupture. Cathepsins are the responsible group of enzymes, some of which are proteinases that shorten proteins to peptides and others that are carboxypeptidases and aminopeptidases that cause the breakdown of peptides to individual amino acids.
The breakdown of tissue caused by bacterial action, which frequently results in the formation of gas. This is known as post-mortem decomposition and is most commonly seen in the intestinal tract, which has a high bacterial content.
Both changes occur quickly after the cell dies, but this takes a variable amount of time after the cell is removed from the body. Cells in the central nervous system die quickly, but others live for a long time.
In general, however, the best results are obtained by putting tissues into fixative as soon as possible after death. If this is not possible, they should be kept in the refrigerator, which slows both autolysis and putrefaction.
It is critical to preserve cells and tissue constituents in as lifelike a manner as possible.
Fixatives’ hardening effect will allow for easy manipulation of naturally soft tissues (for example, brain)
Solidification of Colloid Material
Fixation changes the normal semi-fluid consistency of cells (sol) to an irreversible semi-solid consistency (gel).
Fixation modifies the refractive indices of various cell and tissue components to varying degrees, allowing unstained components to be seen more easily than when unfixed.
Effect on Staining
Most fixatives have an effect on histochemical reactions to some extent because, as previously demonstrated, they generally bind to reactive groups in the tissue to achieve fixation. They may also have an effect on routine staining, sometimes negatively and sometimes positively (picric acid with trichrome stains).
Osmosis and Fixation
It seems obvious that unless fixatives are isotonic solutions, cells will become distorted prior to fixation. Baker (1933) demonstrated that this is not always the case; in fact, under certain conditions, cells reverse their expected behavior. Tissue soaked in 5% acetic acid swells, despite the fact that acetic acid has a pressure of 20 atmospheres, which is roughly three times that of mammalian blood and would therefore be expected to shrink tissues. Saturated aqueous picric acid has a pressure of 2Vi atmospheres, which is roughly one-third the pressure of mammalian blood and should swell tissue, but it shrinks as it fixes. As a result, it appears that the cause of the volume change cannot be osmotic pressure. Fixatives that are protein precipitants shrink tissues regardless of osmotic pressure, whereas non-protein precipitants do the opposite.
Young (1925) demonstrated, however, that certain slowly penetrating fixatives (for example, formaldehyde) must have incorporated an indifferent salt with them to provide an osmotic pressure similar to that of the cell. When distilled water is used instead of saline solution, cells vacuolate and swell. According to Young, the diluent penetrates faster than the fixative, and if the diluent is distilled water, which is hypotonic, it will cause swelling and vacuolation of the cells before the fixative has penetrated. This is probably due to the varying rates of penetration of fixing substances, as well as the temperature and conditions under which they are used.
In practice, the osmotic pressures only need to be close to those of the tissues. There is no benefit to knowing the exact pressure, and there is no benefit to using a complex physiological saline solution when 0.85—0.9% sodium chloride or 1% calcium chloride work equally well.
Loss of Materials during Fixation
Recently, there has been a greater emphasis placed on quantifying the loss of specific entities during fixation as an indicator of their efficiency. It should be emphasized that such papers should be read with extreme caution, especially when it comes to the methods used in the investigation.
As shown below, there is a significant disparity between the results obtained in different laboratories, even if they are of equal renown. This disparity is frequently caused by the fact that, while the experiments themselves are scientifically sound, they do not represent real-world situations. Flitney (1965), using a model instead of tissue, discovered a loss of 76% of protein after formalin fixation, whereas Jozsa and Szederkyi (1967), also using formalin, discovered a loss of 70% of mucopolysaccharides from tissue; however, they washed their tissue in water immediately after fixation. After fixation in formolcalcium, tissues in most routine and research laboratories are processed through graded alcohols, which has been shown by Culling and colleagues (1971) to act as a post-fixation process. They are then cleared and impregnated with paraffin (or Paraplast) for 2—4 hours at 60° C. The effect of heat in this process should not be overlooked, as 4 hours in wax at this temperature will coagulate egg albumen.
Osterowski (1961) investigated this aspect of fixation and discovered that the loss of protein was nil with formaldehyde, 1.4% with Carnoy’s fluid, 8.3% with ethanol, 13% with acetone, and 32-29 percent with freezing techniques. Merriam (1958) found a 6% loss from liver and 4% loss from muscle after formaldehyde fixation, whereas Flitney (1965) found a 76% loss of protein after 2 hours fixation in 4% formaldehyde compared to 11% for 6% glutaraldehyde; however, he was using an artificial albumin-gelatin model, which presumably accounts for such high losses; Hopwood (1968) noted the efficiency of both glutaraldehy Protein loss after osmium tetroxide is well documented (Dallam, 1957; Amsterdam and Schramm, 1966).
Roozemond (1967) reported a 50% lipid loss from the hypothalamus due to water, which was significantly reduced by fixation in formolcalcium. Deierkauf and Hesling (1962) demonstrated that formol calcium fixation did not interfere with subsequent lipid extraction with a chloroform:methanol mixture, and Urbanova and Adams (1970) used an acetone extraction method and noted the preservation of 80% or more of phospholipids after formol calcium fixation. Dermer (1968) demonstrated that there was a 16% loss of lipid even after fixation in osmium tetroxide (which was mostly neutral or phospholipid).
Mucosubstances and Carbohydrates
Tock (1966) demonstrated that freeze drying followed by formaldehyde vapour provided excellent mucin preservation. When Kugler and Wilkinson (1964) investigated the effect of fixation on glycogen, they discovered that the best fixative was ice-cold 80% alcohol, which was confirmed by Hopwood (1967), who also found that formaldehyde preserved glycogen at a rate of 75% and glutaraldehyde preserved glycogen at a rate of 65%.
Using 1 gram pieces of aorta, skin, and cartilage, Jozsa and Szederkyi (1967) discovered that the losses of acid mucopolysaccharides were 60—70% with formol-calcium, 2—3% with formol/cetyl pyridinium bromide, 8% with ethanol, 16—27% with Rossman’s fluid, and 10%—20% with Carnoy’s fluid. They did, however, immediately wash with water after fixation.
Schneider and Schneider (1967) discovered that human cerebral tissue lost DNA, RNA, protein, and lipoprotein after fixation in Carnoy’s fluid or formaldehyde.
Substances with a Low Molecular Weight
Purines, phosphate esters, carbohydrates and indoles (Schneider and Schneider, 1967), catecholamines (Coupland and Hopwood, 1966; Hempel, 1965), ATP and proteins are examples of low molecular weight substances that may be lost in significant quantities (Hopwood, 1968).
Bahr, Bloom, and Friberg (1957) investigated changes in volume and weight in 600 tissue specimens after formaldehyde or osmium tetroxide fixation, processing, and embedding in methacrylate or paraffin wax. In both cases, there was swelling in the fixative; in formalin, it was 9% in 30 minutes and 18% in 12 hours, whereas in osmium tetroxide, the tissue swelled 15% in 15 minutes and 30% in 24 hours. Subsequent dehydration in ethanol resulted in shrinkage (HCHO – 33%, Os04 – 23%); it is worth noting that increasing the number of steps in dehydration resulted in less abrupt changes but with little difference in the end-result. Several dehydrants were used, with methanol being the only one that outperformed ethanol. Infiltration resulted in additional shrinkage of the order of 20% with methacrylate and 30% with paraffin wax. According to the data presented, the final result of formalin fixation and paraffin processing is a total shrinkage of the order of 30-40%. The addition of dextran or another colloid osmotically active substance, according to the authors, reduced these volume changes. Glutaraldehyde alone caused tissue shrinkage (Hopwood, 1967).
The mechanism causing these volume changes is unknown, but it is thought to be related to a number of factors, including changes in membrane permeability, inhibition of respiration, and changes in sodium transport activity.
Rates of Penetration of Fixatives
Dempster (1960) investigated the rates of penetration of nine standard fixing reagents when used alone or in mixtures prepared from standard formulae in a variety of animal tissues (liver, heart, muscle, kidney, brain, and so on), with the depth of penetration measured at various time intervals. The behavior of the standard fixing formulae was of particular interest because it was discovered that mixture ingredients separated out during penetration, with each proceeding at a rate comparable to that of the simple ingredient alone. Dempster demonstrated that after 8 hours in Zenker’s fluid, the bichromate had penetrated rabbit liver to a depth of 0.85 mm, the mercuric chloride had penetrated to a depth of 2.6 mm, and the acetic acid had penetrated to a depth of 5.8 mm. The implication is that the most rapid penetrant in a fixation mixture will have (?) fixed the tissue, with the other ingredients acting as post-fixatives (reacting only with those groups unaffected by the original or most rapid fixative), or that reactions between the secondary fixing agent and the fixed tissue constituents (tissue 4- fixative) may occur, which may be difficult to define at a chemical level.
Flitney (1966) added a complication by demonstrating that the rate of fixation may be affected by the rate of reaction between fixative and tissue in addition to the rate of diffusion. Predicting the chemical reactions involved in complex mixtures of fixing reagents will obviously be difficult. Dempster tested nine standard fixing reagents on rabbit liver, and the results are shown in Table below. The figures for factor ‘K’ (constant) are simply the number of hours required for the first millimeter of tissue to be penetrated; factor ‘e’ is an exponent relating to the rate of diffusion. When factor V (exponent) is close to the square (e=2), the approximate time for complete penetration can be calculated by squaring the required depth in millimeters (half the maximum tissue thickness) and multiplying this factor by the value of the constant ‘K’ (this would apply to fixatives 1, 2, 4, and 7 in Table below).
|Depth of penetration (mm)|
|4 hours||8 hours||12 hours||K||e|
|1||10% Acetic acid||3-8||5||5||0-25||2-11|
|2||5% Trichloracetic acid (c)*||2-7||4||5||0.67||1-78|
|5||7-5% Mercuric chloride (c)||2-0||3-0||3.5||0-79||2-09|
|6||Sat. Aqueous picric acid (c)||1-0||1-5||1-75||3-9||2-03|
|7||2-5% Pot. bichromate||1-0||1-5||1-75||3-94||2-12|
|8||0-7% Chromic acid||0-6||1-0||1-2||8-4||1-99|
|9||4% Osmium tetroxide||0-3||0-5||0-7||23-9||1-94|
Dempster demonstrated the full equation to be t = K.de, where t = time and d = depth penetrated, and factors K and e are as stated above. With a piece of tissue 20 X 10X6 mm to be fixed in formalin (K = 1.14, e = 1.25), an example of using this equation would be t = 1.14 X 31 .2 5 = 4.275 hours.
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