Effect on Tissue Volume of Various Methods of Fixation, Dehydration, and Embedding

A new indirect method is described for following volume changes of homogeneous pieces of tissue during fixation, dehydration and embedding, and area changes during sectioning, staining and mounting. Pieces of rabbit kidney cortex were compared after fixation in Destin's, Orth's, Petrunkevitch's cupric-paranitrophenol, Bouin's, SUSA, Zenker-formol, 10% formalin in distilled water, formalin in saline, Burke's pyridine formalin, CaCOy neutralized formalin, MgCO₃-neutralized formalin, Bensley's vacuum distilled neutral formalin in distilled water, and Bensley's neutral formalin in saline; during dehydration in ethyl alcohol, dioxan, and tertiary butyl alcohol and clearing in xylol and chloroform; and after infiltration and embedding with parowax, with paraffin-nitrocellulose double embedding technic, with hot nitrocellulose, and with cold nitrocellulose. The H-ion concentration of these fixatives was followed during tissue fixation.

Altho all fixatives showed specific merences, SUSA and Bouin's gave the best general results and neutral formalin mixtures, especially pyridine-formalin, the poorest. Isotonic saline was found superior to distilled water as a formalin diluent, reducing tissue swelling during formalin fixation. Reagents producing marked decreases in tissue volume render such tissues less susceptible to shrinkage during subsequent procedures. Shrinkage of tissue during dehydration and infiltration with hot parffin may exceed that produced by fixation alone. Excessive heat causes tissue distortion and shrinkage. Inijltration with hot paran causes considerable shrinkage, with hot nitrocellulose Iess, and with cold nitrocellulose the least sbrinkage.     

A Detailed Analysis of the Effects of Various Fixatives on Animal Tissue with Particular Reference to Muscle Tissue

Nine different fixatives (Carnoy's, Susa, Baker's formalin, 5% formalin, 10% formalin, 10% formol saline, Bouin, Zenker, and 2.5% glutaraldehyde) were compared by two methods. Gelatin-albumin gels were used to study volum changes after fixation and after various stages of subsequent processing. The appearance and hardness of the gels were also noted. The fixatives either shrunk or swelled the gels, but dehydration and clearing shrunk the gels in all cases. Sampkes of muscle tissue from one location in beef longissimus dorsi muscle were also placed in the different fixatives and processed. Various features were noted for each fixative, including the ease with which the paraffin wax blocks were cut and the staining ability of the sections in Mallory's triple stain. The diameters of the muscle fibers were measured from transverse sections of these samples and compared with the mean diameter of muscle fibera in a frozen unfixed section of muscle tissue. It was found that the fixatives had the same shrinkage effects on both the gels and the muscle samples. Analysis of variance tests showed that the various fuatives caused different degrees of shrinkage. Statistical details are given for the amounts of shrinkage caused by each fixative. Both the general histological picture and the amount of shrinkage were considered when deciding the bcst fixative. Carnoy was found to be the best of the fixatives investigated.     

Caliber and media thickness of intrahepatic arteries in a normal human liver. A morphometric study

Morphometrical data on the intrahepatic arterial wall were studied over a wide range of vascular diameters (100-1708 microns) in one human liver. The liver tissue containing Araldite-filled blood vessels was embedded in the water-soluble glycolmethacrylate (GMA). The calculation of the correction factor for the morphometric data obtained from 3 microns sections is described. The influence of formaldehyde fixation on the volume of the liver proves to be negligible. During dehydration a linear shrinkage of about 3% occurs. After infiltration and embedding in GMA only 1% of this shrinkage remains. During the drying phase about 4.3% further linear stretching occurs. No significant "residual compression factor" was found. Araldite plays a significant role in the retention of the dimensions of the liver specimen during histologic processing, while the dimensional changes of the Araldite itself are negligible. A positive linear correlation was found between the media thickness and the radius of the vessel. The physiological consequences are discussed. It is concluded that in the morphometric analysis of the arterial wall it is essential to apply a standardized procedure in histologic processing and in the measuring of the inner vascular diameter. The advantages of our method, in which blood vessels are perfused with Araldite under physiological pressure, are discussed.     

Normal Butyl Alcohol Technic for Animal Tissues with Special Reference to Insects

N-butyl alcohol is substituted in dehydration for the higher ethyl alcohols. No special clearing is necessary as n-butyl is miscible with paraffin.

The greatest advantage of this method is the elimination of both hardening agents (the higher percentage ethyl alcohols and xylol or benzol). Another advantage is the great time toleration of the processes of dehydration and infiltration. For example, tissues have been kept without deleterious effects in n-butyl alcohol for a year before infiltration. Also, aphids which have been kept in a hot (58°C.) paraffin bath for as long as four weeks, have sectioned well. For small insects and vertebrate tissues about five days proved necessary to insure satisfactory infiltration.

N-butyl alcohol was found to give better results than many other technics in serial sectioning of lightly chitinized insects, and in the preparation of embryological and other vertebrate tissues. This technic has been used as a routine method by beginning students in animal microtechnic with better success than attended the usual methods.     

植物材料快速石蜡制片方法

真空干燥箱已越来越广泛地应用于现代生物学研究领域。该文利用真空干燥箱温度和负压的可控制性能,将固定、脱水、透明和石蜡渗透等过程在真空干燥箱中进行,建立起一套可行的植物组织快速石蜡制片方法。结果显示,真空干燥箱的应用加速了多种试剂的渗透速率,提高了切片质量,达到了优化实验步骤、节省实验时间和减少室内有毒化学气体污染的目的。     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Block staining of mammalian tissues with hematoxylin and eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied. General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

A Comparative Study of some Dehydration and Clearing Agents

An experiment to determine the advantages of diozan, iso-butyl alcohol, tertiary butyl alcohol, and ethyl alcohol as dehydrants and chloroform, toluol, xylene, benzol, methyl benzoate, methyl salicylate, and acetone as clearers is described. Materials fixed in Bouin's fluid, Zenker formol, and 10% neutral formalin were dehydrated, embedded, sectioned, and stained. Bouin's fluid produces less hardening, shrinkage and distortion than the other fixatives employed. Slow dioxan is the best method of dehydration. All the picric acid need not be removed from tissues to be embedded in paraffin. Tissue blocks not more than 4 mm. thick may be dehydrated and impregnated with paraffin by slow dioxan in 13 hours, fast dioxan in 10 hours, iso-butyl alcohol and tertiary butyl alcohol in 14 hours, and ethyl alcohol-chloroform in 17 hours without incurring any distortion due to rapidity of dehydration and infiltration.     

Comparative Study of Dehydration

The paraffin method has frequently been criticised because of its hardening and shrinking effect on tissue. The author believes this distortion is due to the dehydration and not to the immersion in melted paraffin. An experimentally controlled series of various tissues was dehydrated in different dehydrating reagents, dioxan, isobutyl alcohol, and ethyl alcohol with chloroform. Except for the dehydration, the tissues were treated identically. In every case, dioxan proved to be a better dehydrating reagent with less shrinkage and brittleness than any of the others. Ethyl alcohol with chloroform produced the greatest degree of distortion.     

The Use of N-Butyl Alcohol in the Paraffin Method

Several modifications in the use of n-butyl alcohol are suggested. These modifications include a revised series of dehydration solutions for exacting work, an abbreviated schedule of limited usefulness, and a simple method for more rapid paraffin infiltration. The use of a triangular coordinate graph may be valuable in designing dehydration procedures for special purposes. Changes in the primary fixation image are significantly less severe by dehydration with butyl alcohol than with many other reagents. Such deleterious effects may be further minimized by reducing the time and temperature factors to the practical limit and by substituting acetone for ethyl alcohol in a dehydration series such as that of Zirkle.     

Frozen Sections from Bouin-Fixed Material in Histopathology

It has become almost a dogma that, for obtaining satisfactory frozen sections, fixation of the tissues in formalin is necessary; formalin is exclusively recommended for this purpose in the textbooks of microscopical and pathological technic.¹ It has, however, several important disadvantages, which become the more acutely felt when fixation in more than one fixative is impossible, as happens so often in surgical pathology. When, after the preparation of the frozen sections, embedding in paraffin is necessary, formalin-fixed tissues show a marked shrinkage, especially when rapid embedding methods (e. g. dioxane) are used. This shrinkage can only be prevented by the time-consuming hardening of the blocks in K₂Cr₂O₇. The trichrome stains, the phosphotungstic-hematoxylin stain and the azan method, which are slowly superseding the hematoxylin-eosin and van Gieson stains in histopathology, give only mediocre or inferior results after formalin fixation, unless the sections are refixed.     

The Use of N-Butyl Alcohol in the Paraffin Method

Several modifications in the use of n-butyl alcohol are suggested. These modifications include a revised series of dehydration solutions for exacting work, an abbreviated schedule of limited usefulness, and a simple method for more rapid paraffin infiltration. The use of a triangular coordinate graph may be valuable in designing dehydration procedures for special purposes. Changes in the primary fixation image are significantly less severe by dehydration with butyl alcohol than with many other reagents. Such deleterious effects may be further minimized by reducing the time and temperature factors to the practical limit and by substituting acetone for ethyl alcohol in a dehydration series such as that of Zirkle.     

Processing of immunoisolated pancreatic islets: implications for histological analyses of hydrated tissue

Routine tissue processing is usually associated with histological artifacts as a consequence of shrinkage and distortion during dehydration required for embedding. With hydrated specimens such as lung, embryonic, and tissues in hydrophilic membranes, tissue processing can induce severe artifacts that interfere with adequate microscopic evaluation. Here we present a method for embedding hydrophilic alginate-polylysine microencapsulated pancreatic tissue that combines the absence of histological artifacts with a practical tissue processing method. We found that the glycol-methacrylate (GMA)-embedding method preserved the integrity of the encapsulated tissue better than snap-freezing or paraffin embedding, but the overall quality of the hydrophilic capsules remained poor Next, we modified the GMA method by introducing gradual dehydration to investigate whether the integrity of the sectioned capsules was better maintained by a more gradual pattern of water extraction. This modification resulted in well-preserved morphological details of the hydrophilic membranes, hydrogel-cell interface, and encapsulated pancreatic tissue. Subsequent routine staining gave excellent contrast between the islet tissue and hydrophilic components, which allowed adequate quantitative histological and pathological comparisons.     

Protein fixation and antigen retrieval: chemical studies

Abstract

Fixation with formaldehyde is the first process to which most biopsy and necropsy specimens are exposed prior to dehydration and embedding in paraffin wax. Tissue specimens that have been fixed in formaldehyde have architectural characteristics that are familiar to virtually every pathologist and these facilitate routine diagnosis. Nevertheless, formaldehyde fixation has some deleterious effects including reduction in immunoreactivity and degradation of nucleic acids. Development of methods to counteract these deleterious effects requires an understanding of the chemical events that occur during tissue fixation and subsequent tissue processing. This short review illustrates some of the chemical consequences of formaldehyde fixation and ethanol dehydration. It also provides some insight into the molecular events accompanying heat-induced antigen retrieval.     

Determination of lipid loss during aqueous and phase partition fixation using formalin and glutaraldehyde

Phase partition fixation permits fixation of tissue in a nonaqueous environment, thus eliminating osmotic effects. It was shown in an earlier investigation that retention of protein in liver blocks can be improved by phase partition fixation. By using radioisotopic labeling techniques, the effects of phase partition fixation on lipid retention during fixation, dehydration, and clearing have been determined and compared with those of standard aqueous fixation techniques. In this article we show that retention of total lipid in liver blocks following phase partition fixation using formalin was comparable to or better than that with aqueous formalin fixation and processing. Fixation with glutaraldehyde using phase partition fixation resulted in somewhat greater loss of total lipid than that observed for aqueous buffered glutaraldehyde-fixed blocks.     

A Modified Freezing-Drying Apparatus for Tissues

The preparation of tissues by the freezing-drying technic is preferred for many histochemical studies because of the rapid fixation, avoidance of deleterious action of chemical fixation and extraction by aqueous and lipid solvents in fixation, dehydration and clearing; to facilitate this procedure a freezing-drying apparatus has been constructed which permits cutting of sections within five hours after the fresh tissue is obtained. Liquid nitrogen provides a most efficient moisture trap and in conjunction with a heating element provides any desired temperature down to — 80°C. during tissue drying. Paraffin infiltration is started without disassembling the equipment and completed in a few minutes. Most stains and histochemical reactions for enzyme and other substances can be applied directly to sections of frozen-dried tissue cut from the same blocks.     

Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.     

Stain for histamine in mast cells in fixed tissue

Summary A technique is described for staining histamine in mast cells of tissues fixed in alcohol. The method employs cold ethanol fixation, xylene clearing, paraffin embedding, and staining of the sections with 1% o-phthalaldehyde in ethyl benzene in a humid chamber. This study was supported by a grant from the John A. Hartford Foundation.     

Immunohistologic techniques for detecting the glycolipid Gb3 in the mouse kidney and nervous system

Shiga toxin-producing Escherichia coli causes hemolytic uremic syndrome, a constellation of disorders that includes kidney failure and central nervous system dysfunction. Shiga toxin binds the amphipathic, membrane-bound glycolipid globotriaosylceramide (Gb(3)) and uses it to enter host cells and ultimately cause cell death. Thus, cell types that express Gb(3) in target tissues should be recognized. The objective of this study was to determine whether immunohistologic detection of Gb(3) was affected by the method of tissue preparation. Tissue preparation included variations in fixation (immersion or perfusion) and processing (paraffin or frozen) steps; paraffin processing employed different dehydration solvents (acetone or ethanol). Perfusion-fixation in combination with frozen sections or acetone-dehydrated tissue for paraffin sections resulted in specific recognition of Gb(3) using immunohistochemical or immunofluorescent methods. In the mouse tissues studied, Gb(3) was associated with tubules in the kidney and neurons in the nervous system. On the other hand, Gb(3) localization to endothelial cells was determined to be an artifact generated due to immersion-fixation or tissue dehydration with ethanol. This finding was corroborated by glycolipid profiles from tissue subjected to dehydration; namely Gb(3) was subject to extraction by ethanol more than acetone during tissue dehydration. The results of this study show that tissue preparation is crucial to the persistence and preservation of the glycolipid Gb(3) in mouse tissue. These methods may serve as a basis for determining the localization of other amphipathic glycolipids in tissue.     

Method for the Prfservation of Polystyrene Latex Beads in Tissue Sections

The degree of infiltration of epoxy resin into pituitary secretory granules was evaluated using X-ray microanalysis of the concentrations of chlorine in the epoxy resins. The effectiveness of infiltration was tested after three different tissue preparation techniques: cryofixation + freeze-drying (CF-FD), glutaraldehyde fixation (GF) + chemical dehydration, and no fixation— no dehydration. Signs of marked incomplete infiltration were found in embedded unfixed tissue while the other two techniques showed 80% infiltration. Uneven penetration was seen after CF-FD and GF. The plastic surface demonstrated a mountain-like appearance over the secretory granules after immunocytochemistry of the glutaraldehyde fixed tissue, whereas the CF-FD tissue showed a less furrowed surface. This probably is due to contact with water, which swells those parts of the granules that are unprotected by the plastic embedding medium. Our findings may explain why it is possible to perform immunocytochemistry on Epon embedded tissue.