Histochemical Demonstration of Enzyme Activities in Plastic and Paraffin Embedded Tissue Sections

Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, β-galactosidase, and cytochrome oxidase in plastic embedded and routine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 μm, were far superior to frozen Sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.     

Paraffin Sections of Tissue Fragments

Pathologists are frequently called upon to examine minute fragments of tissue obtained by aspiration or other similar means. Although the smear technic is generally satisfactory for this purpose, there are occasions when sections are preferable. This is the case when study of the interrelationship of cells is desired, or when one wishes to avoid the distortion produced in cells by smearing.     

Staining Paraffin Embedded Sections of Scald of Barley before Paraffin Removal

Staining of paraffin embedded sections with periodic acid-Schiff reagent and fast green before paraffin removal resulted in differentiation of barley seed and leaf tissue from fungal structures of Rhynchosporium secalis. Crystal violet, toluidine blue O and aniline blue also successfully stained fungal structures of R. secalis in barley leaf tissues. Staining of embedded sections before paraffin removal allows simple processing of a series of sections, saves time and reduces solvent consumption.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

A Method for Processing Paraffin Sections of Multilayer Cultured Tissue

A simple and rapid method is described for processing histological preparations from multilayer cultures growing in plastic Petri dishes. A covering collodion film is utilized to remove the tissue from the plastic dish and transfer it onto a paper block prior to embedding in Paraplast. To avoid any disruption by the collodion of the plasticware, the cultured tissue is first immersed in a solution of collodion and absolute alcohol (1:1) and then covered with pure collodion. All steps are carried out in the cold. This procedure allows morphological, histochemical, immunofluorescent, and autoradiographic studies to be carried out on serial sections of cultured tissue.     

A Method of Affixing Separate Thick Sections from Materials Embedded in Paraffin

Botanical studies often require thick histological sections (for embryology, pollen and spore arrangement in tetrads, etc.). Study of the original position of the generative cell in Angiosperms, for example (Huynh 1972), requires paraffin sections bearing entire pollen grains with a diameter of up to 80 μm. However, it is impossible to obtain ribbons with sections of such thickness. If the sections are affixed separately, they do not hold so strongly to slides as do those mounted as ribbons; this difficulty increases with thickness of section. in addition, affixing sections separately with the required order and spacing is tedious and difficult, demands a great deal of time, and even so, is not always successful. the simple method described here can remedy such inconveniences.     

A Simple Procedure to Obtain One-Micrometer Sections of Routinely Embedded Paraffin Material

By freezing blocks of paraffin-embedded tissues to a convenient temperature it is possible to obtain routinely 1 µm sections that can be further processed as normal thicker sections. Normal and disposable steel knives can be used and the staining time should be increased in most procedures. Gradual freezing of blocks to the temperature of dry ice is the simplest and safest way to obtain an adequate temperature. The best results were obtained using as fixative 4% paraformaldehyde in phosphate buffered saline solution.     

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using ''filter aided sample preparation'' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on ''pipette-tip'' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.     

Fluorescence of Plastic Embedded Tissue Sections After Curcumin Staining

The natural dye, curcumin (C.I. 75300) from turmeric, is obtained from the roots of Curcuma longa (Lillie 1977). Curcumin has scarcely been applied for histological work, and its fluorescence seems to have been overlooked. During the course of studies on fluorescent aluminum complexes (Del Castillo et al. 1987) we realized that this dye induces a green fluorescence of chromatin (Stockert et al. 1989). In this note we describe the fluorescence reaction of curcumin on semithin sections of olastic embedded tissues.     

In Situ Hybridization: Use of S-Labeled Probes on Paraffin Tissue Sections

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with ³⁵S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2–3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.     

Tissue Shrinkage Caused by Attachment of Paraffin Sections to Slides: its Effects on Staining

Sections were cut from a wide variety of tissues, and those from each block were divided into four groups before attaching and drying on slides. Four commonly accepted sources of heat were used for drying: (a) gas hotplate set at 65° C; (b) incubator, 37°; (c) oven, 56°; and (d) room temperature, 20°. After drying, the sections were stained, then examined for intensity of staining and for distortion caused by shrinkage. With both soft and decalcified tissue stained by haematoxylin and eosin, the best results occurred in the sections dried at 20° C; the next best at 37°. When stained by Van Gieson's method, both types of tissues were best after 20° drying, but the second-best group showed differences in favour of 56° for soft tissues and 37° for decalcified. After drying decalcified tissue at 65°, the staining of collagen by acid fuchsin was almost completely absent. When impregnated with silver, for reticulin, the best results for soft tissues were after 56° drying; second best, 20°; but decalcified tissues showed a reversal of this order. After PAS, there was an increasing intensity of staining from 20° to 65°, with soft tissue; evidence that histochemical interpretation could be strongly influenced by drying temperature.     

Cutting Frozen Sections on a Paraffin Microtome

Into a hole drilled in a block of dry ice, a metal microtome object disk is placed to cool. A drop of water is placed on the disk, and the specimen to be cut is fixed in place. By setting the dry ice in a well-insulated box, the specimen is thoroughly frozen. The disk is then clamped in the microtome, and chips of dry ice are wedged between the metal disk and the object clamp of the microtome. This ensures the continued cooling of the specimen while the tissue is being cut.     

A Hematoxylin and Eosin-Like Stain for Glycol Methacrylate Embedded Tissue Sections

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5 percent ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075 percent ponceau de xylidine and 0.025 percent acid fuchsin in 10 percent acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.     

A Gelatin Fixative for Paraffin Sections

Mayer's albumen fixative, of which the active principle is white of egg, is used almost universally for affixing paraffin ribbons to the slide. About eight years ago the writer's attention was called to a gelatin fixative which has proved to be so superior to albumen that he has used it almost exclusively ever since in the making of a great variety of botanical preparations, and has recommended it to a number of other workers whose experience with it subsequently has been just as satisfactory. The gelatin method was first described by Szombathy¹ and later discussed by Artschwager,² but it does not seem to have received the attention in the literature which its importance deserves. It certainly merits a wide spread use among both botanists and zoologists.     

Acridine Orange Fluorescence in Paraffin Sections

The technique of staining with acridine orange for fluorescence microscopy of fresh animal and plant cells, chiefly for the detection of ribonucleic acid in the cytoplasm, was brought to a high degree of perfection by Schümmelfeder (1950) and has been developed further by Bertalanffy and Bickis (1956). Its employment for cancer detection in smears was reviewed by Bertalanffy, Masin and Masin in 1956.     

A Gelatin Fixative for Paraffin Sections

Mayer's albumen fixative, of which the active principle is white of egg, is used almost universally for affixing paraffin ribbons to the slide. About eight years ago the writer's attention was called to a gelatin fixative which has proved to be so superior to albumen that he has used it almost exclusively ever since in the making of a great variety of botanical preparations, and has recommended it to a number of other workers whose experience with it subsequently has been just as satisfactory. The gelatin method was first described by Szombathy¹ and later discussed by Artschwager,² but it does not seem to have received the attention in the literature which its importance deserves. It certainly merits a wide spread use among both botanists and zoologists.     

Postadhesive Technique for Archival Paraffin Sections

We present a postadhesive protocol for adhering paraffin sections of archival material to microscope slides. Appropriately posttreated sections, subsequently processed for immunohistochemistry, remained attached to the slides and were well preserved with no signs of artifacts, such as scratching and shrinkage. The immunohistochemical staining was intense and antigen-specific without nonspecific background. Specific staining intensity was equal to that produced in untreated control sections; however, the latter became partially or fully detached from the slides. The postadhesion protocol may be used with modern techniques and is recommended for reclaiming use of otherwise unsuitable paraffin sections of archival material.     

Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography

Background

Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data.

Methods

FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging.

Results

The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections.

Conclusion

We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.     

植物冰冻切片条件的优化及其与石蜡切片在组织化学应用中的比较

冰冻切片是植物组织学研究中一项重要的实验技术,冷冻温度和冷冻时间是决定切片质量的关键因素。通过比较15种冰冻切片条件,得出植物组织直接冰冻切片较适宜的冷箱温度、冷台温度和冷冻时间。同时,通过对5种植物的不同组织进行组织化学染色,比较了新鲜材料直接冰冻切片与常规石蜡切片在不同化学成分鉴定上的异同及各自的适用范围。结果表明,对于多糖、蛋白质和角质,两种切片方法的鉴定结果比较一致,但对于脂肪只能采用冰冻切片技术。研究结果对植物组织学实验和研究方法的改进具有一定的参考价值。