Plant Cuticle Staining with Bismarck Brown Y and Azure B or Toluidine Blue O before Paraffin Extraction

Transverse paraffin sections of mature greenwood stems of rose (Rosa x hybrida) and flowering dogwood (Cornus florida L.) were stained with Bismarck brown followed by azure B or tolnidine blue O. The Bismarck brown was replaced by thiazin dye metachromasia in all structures except the cuticle which remained brown or yellow. The interface between the cuticle and exterior cell walls of the epidermis was delineated clearly.     

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.     

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.     

A Clearing Technique for Detecting the Fungal Endophyte Acremonium sp. in Grasses

Leaf tissue of tall fescue Festuca arundinacea Schreb., hard fescue Festuca ovina L., red fescue Festuca rubra L. and perennial ryegrass Lolium perenne L. was stained with rose Bengal or aniline blue to detect the presence of the fungal endophyte Acremonium sp., Specimens were cleared using methyl salicylate, an optical clearing agent, and viewed using bright field microscopy. Tissue was presenred as dried tissue or stored in 70% aqueous ethyl alcohol before staining and clearing. Tissue was observed at 2, 4 and 12 weeks following clearing to check for stain retention. Staining with rose Bengal was inferior to aniline blue when followed by the clearing agent methyl salicylate. Fungal mycelia stained lighter with rose Bengal and were more difficult to detect than mycelia stained with aniline blue. The results illustrate the usefulness of combining staining and methyl salicylate clearing for detecting fungal endophytes.     

Modified Tetrachrome Method for Osteoid and Defectively Mineralized Bone in Paraffin Sections

A new modification of the tetrachrome method for bone osteoid in paraffin sections has been designed. The modified tetrachrome method suitable for routine use in any histology laboratory retains the simplicity of the original method and gives good results on the freshly fixed, decalcified, paraffin embedded material. Osteoid tissue is stained deep blue and normally mineralized bone is stained red. Defectively mineralized bone stains pale blue or pink and the cellular population is clearly identifiable. The ability to distinguish the osteoid tissue from mineralized bone and connective tissue and cartilage makes diagnosis of osteomalacia or osteoid producing tumors or assessment of ossification process straightforward, without the need for un-decalcified sections. By displaying simultaneously irregularities in the mineralized matrix and morphology of bone cells, the method also permits the diagnosis of conditions recently described in patients with osteoporotic fractures, such as osteocytic degeneration and bone tissue defects.     

Novel Fixation of Plant Tissue, Staining through Paraffin with Alcian Blue and Hematoxylin, and Improved Slide Preparation

Onion (Allium cepa) root tips were fixed in a proprietary solution without aldehyde, toxic metals or acetic acid. Fixed specimens were embedded in paraffin, sectioned on a rotary microtome and mounted on detergent-washed slides without adhesive. Slides with ribbon segments affixed were immersed in 0.2% aqueous alcian blue 8GX in screw-capped Coplin jars in a water bath at 50 C for 1 hr. Excess alcian blue was rinsed off under cold running tap water and the slides were immersed in quick-mixed hematoxylin at room temperature for 15 min. Stained slides were deparaffinized, rinsed with isopropanol, air dried, and coverslips were affixed with resin. Thus, the traditional paraffin microtechnique has been modified at all steps from fixation to finishing slides with coverslips.     

The Demonstration of Beryllium Oxide in Paraffin Sections with Chromoxane Stains

Aqueous solutions of the arylmethane dyes Chromoxane pure blue BLD (C.I. No. 43825) and Chromoxane pure blue B (C.I. No. 43830) will stain beryllium oxide. In the presence of EDTA the staining of other metals is masked. As a specific stain for BeO, formol saline fixed paraffin sections are hydrated and stained for 1 hr with either 0.1 gm of pure blue BLD in 100 ml of pH 4.0 Na-acetate buffer or with 0.1 gm of pure blue B in 1 N NaOH adjusted to pH 9.0 with HCl. To mask interference from other metal ions, 9 gm of Na₂-EDTA is added to 100 ml of the stain solution. BeO is stained blue, organic tissue components are either unstained or pink. Results of tests against other materials show that a high degree of specificity may be expected from these dyes. A 1% aqueous solution of neutral red may be used as a counterstain.     

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.     

Staining Mycobacterium Leprae in Paraffin Sections by the Gomori Methenamine-Silver Method

Gomori's methenamine-silver method can be used successfully for staining Mycobacterium leprae in paraffin sections even though these sections are obtained from 10-yr-old blocks, in which the acid-fastness of the microorganisms no longer exists.     

Light and Scanning Electron Microscopy of Greenbug Aphid Damage in Wheat Using the Same Section

A method has been developed to enable correlative light microscopy (LM) and scanning electron microscopy (SEM) on the same section of wheat (Triticum aestivum L.) leaves infested by greenbug aphids (Schizaphis gra-minum Rondani). Segments of infested leaf tissue were fixed, embedded in paraffin, sectioned, and affixed to slides by standard histological techniques. Serial sections were viewed by LM as temporary mounts in xylene. Sections of interest were identified and re-embedded in fingernail polish, affixed to aluminum stubs, freed of polish with ethyl acetate or acetone, and sputter-coated for SEM. SEM of re-embedded leaf sections showed excellent preservation of leaf anatomy. The same aphid tracks and regions of cell damage identified by LM were visible. SEM increased resolution and provided a much clearer sense of the three-dimensional relations involved in the interaction between plant and insect.     

Conjugates of Chitinase with Fluorescein Isothiocyanate or Lissamine Rhodamine as Specific Stains for Chitin In Sztu

The fluorescent chitinase technique is based on the specific affinity of the enzyme for its substrate and applicable when an enzyme can be coupled with a fluorescent dye. Fluorescent chitinase specifically stained chitinous structures in several fungi and an insect, but failed to stain other polysaccharides in bacterial and algal cell walls. Freezing-microtome sections of Drosophila and fungal mycelia 6 μ thick were fixed in acetone for 5 min, then stained and mounted in fluorescent chitinase. Staining of smears of unsectioned fungal material required 5 min in absolute acetone, 5 min in 95% ethanol-1 N aqueous acetic acid (1:1), 10 min in 0.2 M phosphate buffer, PH 5.7, 1 sec in enzyme-dye conjugate, and 10 min in carbonate-bicarbonate buffer (0.2 M, pH 10.7, for chitinase-FITC; pH 7.6, for chitinase-LRBC). Preparations are viewed microscopically with ultraviolet light.     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

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。     

The Use of Ethylenediamine in Softening Hard Plant Structures for Paraffin Sectioning

Ethylenediamine has been used as an agent for softening very hard woods prior to sectioning on a sliding microtome. The use of ethylenediamine is recommended for two additional uses: for preparing 1) soft woods in which wide, thin-walled tracheids or vessels tend to collapse during sliding microtome sectioning and 2) plant tissues with sclerenchyma mixed with soft-walled cells (bark, leaves, fruits, etc.) which frequently fail to section well. After softening in ethylenediamine, material is washed, infiltrated, and embedded in paraffin. Preliminary sections are made with a rotary microtome, just exposing the cut surface of the material; this exposed surface is soaked overnight in water. Sectioning is then continued. Sections produced in this fashion are considerably improved. The wood and pith of Podocarpus ustus, a parasitic conifer from New Caledonia, is used as an object to demonstrate improvements in sectioning by the ethylenediamine-paraffin method. Thinner sections with minimal tearing, cell collapse, and unevenness are produced. Sections can be handled easily and stained more effectively than unmounted sections. Variations in timing and in treatment are recommended to suit different materials. Ethylenediamine, used with reasonable caution, is much less hazardous than hydrofluoric acid and is more effective in softening plant material. The ethylenediamine method may be used routinely on any material difficult to section because of hardness.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

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。     

A Methylene Blue-Basic Fuchsin Stain for Mast Cells in Paraffin Sections

In paraffin sections of rat tissue it is possible to stain mast cell granules blue in contrast to red nuclei, pale blue cytoplasmic ribonucleic acid, and colorless collagen. This is done by the following mixture: 1% methylene blue (pure, not polychrome), 9 ml; 0.1% basic fuchsin, 9 ml; glacial acetic acid, 2 ml. Stain formol-fixed, paraffin-processed sections for 5 min, wash in water and pass through acetone, 2 changes, 10 sec total, to xylene and a polystyrene mounting medium.     

A Method for Staining Infection Hyphae in Pine Leaves

Fungus-inoculated Pinus radiata leaves were fixed and then stained with periodic acid-Schiff reagent. Pieces of leaf with fungal material on the surface were removed. These pieces were stained in lactophenol cotton blue for a few minutes and then mounted in dilute lactophenol cotton blue. Microscopic examination of fungal material inside and outside the mounted leaf pieces revealed the following: conidia and germ tubes on the leaf surface were red, appressoria remained unstained, and infection hyphae within the leaf were stained blue. This differential staining method was particularly useful for distinguishing germ tubes from infection hyphae arising from appressoria.     

An Evaluation of the Metachromasy of Anionic Dyes. I. Visual Observations on Tissue Sections

Thirteen dyes of the azo (benzopurpurin, Congo red, trypan blue, chromotrope 2R, orange G), indigoid (indigocarmine), triphenylmethane (acid fuchsin, aniline blue, light green, methyl blue), and xanthene (eosin B, eosin Y, erythrosin B) groups were applied under standard conditions to a variety of human, rabbit, rat, mouse and frog tissues in paraffin sections. Sections were examined for color changes which might indicate metachromatic reactions analogous to the metachromasy of cationic dyes. Disazo and xanthene dyes showed shifts in hue, with some qualification on the shifts of xanthenes. Metachromatic shifts of anionic dyes were generally of low order compared to those of cationic dyes. Nuclei, erythrocytes, inner elastic laminae of arteries, keratinous structures, and certain areas in the ground substance of connective tissue most often elicited metachromasy. It is suggested that basic proteins are responsible for the metachromatic reactions. Equally interesting areas were those staining poorly (cartilage matrix, most types of mucus), since these are sites of highly acidic substances capable of binding proteins.