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.     

Enzyme Histochemistry on Paraffin Embedded Tissue Sections

To obtain diagnostic enzyme reactions in paraffin embedded tissue sections, we compared four fixatives (buffered formol sucrose, Baker's formol calcium, periodate lysin paraformaldehyde, and buffered formalin acetone) and subsequent acetone dehydration with or without graded concentrations of Triton X-100. Four spleens and 14 lymph nodes were tested for peroxidase, naphthol ASD chloroacetate esterase, acid phosphatase, alpha naphthyl acetate esterase, and alpha naphthyl butyrate. Best results were obtained by a processing method using buffered formalin acetone, Holt's gum sucrose, dehydration in acetone with 0.03% Triton X-100, and paraffin for embedding.     

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.     

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.     

Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.     

Application of the Papanicolaou Stain to Paraffin Sections

Routine paraffin sections from tissues fixed either in aqueous formalin, 80% alcohol (with or without 1% trichloracetic acid added), Carnoy's alcohol-chloroform-acetic (6:3:1) and Bouin's fixative were stained as follows: Harris' hematoxylin, 6 min; running water, 2-3 min; ascending grades of alcohol to 95%; orange G, 0.5% and phosphotungstic acid, 0.015% in 95% alcohol, 5 min; 95% alcohol, 2 changes; Papanicolaou's EA36, 2.5 min; dehydration, clearing, and covering in Permount. The results show morphology better than hematoxylin and eosin and the technic is recommended particularly for keratin, which always stains bright orange.     

A Method of Orienting Paraffin Sections for Three-Dimensional Reconstructions

To make reconstructions from serial sections, reference points are needed to orient the sections. Such points can be provided after paraffin embedding by cutting the bottom face of the block to form a plane and adding a groove along the center of this plane. The plane and groove are coated with Mimeograph Correction Fluid and the block is built up by dipping in hot paraffin so that the marked plane lies inside the block. Each section will have a blue line with a notch in it representing the plane and groove. This line remains through staining and is used to orient each section with respect to an eyepiece reticle. The reticle, in effect, supplies X and Y coordinates for every point in the specimen while the number of each section counted from one end is a Z coordinate.     

Staining Paraffin Sections Without Prior Removal of the Wax

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10-20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.     

Serial Sections; Simplified Handling of Paraffin Ribbons on a Floating-Out Bath

Pieces of para & ribbon containing serial sections are arranged in overlapping rows on a microscope slide coated with albumen or glycerol. The assembly of sections is then floated free by immersing the slide in a bath of warm water. The rows of sections forming the assembled unit adhere to each other along their overlapping edges. After the sections have softened and expanded, the unit is picked up on a slide, covered with wet filter paper, rolled flat with a photographic print roller, and allowed to dry.     

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.     

HistoGel预包埋法在微小组织石蜡切片中的应用

目的：探讨微小组织HistoGel预包埋的石蜡切片制作方法和优势。方法：选择拟胚体(embryoid body, EB)为微小组织,将人 类胚胎干细胞(human Embryonic Stem Cell, hESC)切割成小块,用拟胚体培养基在低贴附培养皿中悬浮培养形成拟胚体。收集拟 胚体,4 %的多聚甲醛固定。将HistoGel 加热到60 ℃熔解,离心去除拟胚体中的固定液,把液态的HistoGel 加到拟胚体上,调整拟 胚体的相对位置使其相对集中,待胶冷却凝固,将含有拟胚体的胶块取出,进行常规的石蜡切片操作,包括脱水、透明、浸蜡、包 埋、切片和苏木素- 伊红(hematoxylin-eosin, HE)染色。显微镜下观察拟胚体的形态,并用拟胚体的形态完好度来评判这种方法。结 果：HistoGel 仅在60 ℃便可熔解,室温可以冷却凝固,含有拟胚体的胶块在整个操作过程中很方便。展片过程中,石蜡和HistoGel 能够保持平整。HE 染色的结果可以看出,拟胚体内部结构完好,细胞核和细胞质清晰可辨。结论：HistoGel可以作为一种微量细 胞组织的预包埋胶制作石蜡切片,而且相比琼脂预包埋,HistoGel 因其操作更加方便和特有的物理特性显示出更多的优点。     

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.     

两种脱钙法植被鼠尾病理切片的比较

目的探讨制作大鼠尾巴标本石蜡切片的方法。方法采用盐酸脱钙液运用两种脱钙方法制作大鼠尾巴标本石蜡切片。结果两种方法制作的石蜡切片完整、无破碎,HE染色观察组织结构细胞形态完整,核浆分明,红蓝适度。结论两种方法都能制作理想大鼠尾巴标本石蜡切片,可保证病理诊断质量。     

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.     

Preservation of Soluble Proteins for Histological Staining in Paraffin Sections

Human serum at full strength and in dilutions with physiological saline (0.85%) ranging from 1:1 to 1:72 was allowed to permeate rectangular masses of fibrin foam in small pieces (maximum diameters 0.2 × 0.4 × 1.0 cm), and then placed in 10% neutral formalin, Zenker's solution and Bouin's solution. After fixation for 4-12 hr, the fibrin foam and occluded serum proteins were imbedded, sections cut and stained with eosin bluish (CI. 771), 0.25% alcoholic solution, and by the McManus periodic acid-Schiff technique, using basic fuchsin (CI. 677). Undiluted serum (6.4 gin 100 ml) was not stainable after fixation in 10% formalin. With Zenker's solution stainable serum proteins are recognizable at 0.22 gm/100 ml and with Bouin's solution at 0.08 gm/100 ml. Dried aliquots (0.2 ml) of the same dilutions, spread over an area of 1.0 cm², fixed and stained similarly, gave almost identical results.     

Visualization of Legionella Pneumophila in Paraffin Sections using Hexamine Silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 μm. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.     

Digestion of Collagen and Reticulin in Paraffin Sections by Collagenase

Tissues are fixed in ethanol or in Carnoy's 6:3:1 mixture and embedded in paraffin after routine ethanol dehydration. Sections are taken to water and then covered with 0.2 ml of a 0.9% NaCl solution containing 1 mg/ml of collagenase, and incubated at 50° C for 45 min. After this, they were washed and then stained by the usual methods for connective tissue fibers. Control sections were made by substituting plain 0.9% NaCl solution for the collagenase solution. The collagenase used was from bacteria and obtained from Nutritional Biochemicals Corporation, Cleveland 28, Ohio.     

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.     

Sandwich Embedding of Eyeball Wall for Optimal Paraffin Sections of Retina

Whole human or animal eyeballs are fixed in Heidcnhain's Susa 2-4 hr, the posterior chamber then opened by removing the anterior portion of the bulb and fixation continued for 3-6 hr. Mercurial precipitates are removed by 0.5% iodine in 80% alcohol, 2 changes of 2-4 hr each. Pieces of the bulbar wall, about 3 × 5 mm, are enclosed between two slices of formalin-fixed liver (dehydrated to 80% alcohol), bound with thread, dehydrated, and infiltrated with paraffin. The thread is then removed and the block cast.     

木本植物非均质化组织石蜡切片制作方法

在常规石蜡切片技术的基础上, 针对木本植物茎段木质化程度高、硬度大以及各部分组织硬度不均匀等特点, 选取核桃(Juglans regia)茎段以及芽接愈合区域组织为实验材料, 对固定、软化和脱水等关键步骤进行改进, 获得结构完整且染色清晰的茎段组织和嫁接愈合区域组织切片, 可清晰地观察到各部分组织的形态特征和愈合过程中的发育特征, 且缩短了制片周期。采用改良后的实验流程成功获得了苹果(Malus pumila)、桃(Amygdalus persica)、杏(Armeniaca vulgaris)、李(Prunus salicina)和杨(Populus tomentosa)的茎段横截面切片。该方法为从解剖学上研究林木茎段生长机制和形态发育变化提供技术基础, 为非均质化植物材料的石蜡切片制作提供参考。