An Improved Immunolabeling Method for Microtubular Cytoskeleton in Poplar (Populus nigra L) Free Nuclear Endosperm

We investigated immunocytochemical staining of microtubular cytoskeleton of free nuclear endosperm, a tissue which is particularly difficult to fix. This tissue requires fixation for 45 hr to preserve the integrity of the microtubular network after paraformaldehyde based fixation. Low glutaraldehyde concentration in the fixative and the ethanol dehydration retains β-tubulin antigenicity and the former improves preservation of tissue structure. An ethanol-free embedding method is recommended for immunocytochemical studies of ethanol sensitive target proteins.     

Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.

Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations. To determine the effect of fixation on the preservation of the nucleic acids, we fixed two randomly chosen fresh pathology specimens in formalin, B-5, Bouin's, Zenker's, ethanol, and Omnifix for 6, 24, 48, 72, and 168 hr (1 week), and then embedded the tissue in paraffin. Oligonucleotide primers specific for the cytoplasmic-beta-actin gene were chosen to span an intron such that amplification yielded a product of 250 BP for DNA and 154 BP for RNA. A single 6-microns section was cut from each paraffin block, deparaffinized, and then subjected to 30 rounds of amplification for either DNA or RNA. On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr of fixation time, whereas variable product was seen with formalin or Zenker's fixation; all specimens fixed in Bouin's or B-5 were negative. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr in the formalin-fixed tissue. The Zenker's-fixed tissues gave variable results, and the Bouin's and B-5 tissues gave consistent results only after 6 hr of fixation. We therefore conclude that choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material.     

Ultrastructural patterns of the flagellar axoneme in the non-motile part of the mole-cricket sperm

The mole-cricket spermatozoon (Gryllotalpa gryllotalpa) has a motile anterior tail region and an immotile posterior region. The posterior portion appears stiff and its microtubular doublets and central singlet microtubules are swollen, apparently due to an excess of material within them. In particular, doublet number 6 is of an unusually large size. The general organization of the axoneme is also modified by a loss of dynein arms and spokes in the posterior portion. When studied by a fixation technique that involves tannic acid to outline the protein molecules and PA-TCH-Ag method for staining polysaccharides it could be seen that the microtubular doublets and accessory microtubules contain rounded globules surrounded by polysaccharides. The arrangement of protofilaments within the microtubular walls is visible both in the anterior tail region with normal doublets and in the posterior region with degenerated doublets.     

Preservation of cytoskeletal elements for electron microscopy.

Attached murine fibroblasts were subjected to different fixation and dehydration procedures in order to study the preservation of cytoskeletal elements. The organization of the microtubular and microfilamentous system was identical whether or not post-fixed with osmic acid after glutaraldehyde fixation. Critical point drying did not preserve structures absent in conventionally dehydrated and embedded cells.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective fluorescence of eosinophilic structures in grasshopper and mammalian testis stained with haematoxylin-eosin

After staining with Mayer's haematoxylin and eosin Y, paraffin sections of grasshopper and mouse testis were analysed by both transmitted light and fluorescence microscopy. Under violet-blue (436 nm) light excitation, a bright green emission was observed in all eosinophilic structures. Meiotic spindles (fibres and poles), mitochondrial aggregates, centriolar adjuncts in grasshopper spermatids, the basal lamina, flagellar bundles and remaining cytoplasmic droplets in the lumen of seminiferous tubules showed the most striking fluorescence induced by eosin Y. No emission was found in these structures after haemalum staining. Fluorescent microtubular components also revealed a positive immunoperoxidase reaction for -tubulin. All fixation and embedding procedures (Bouin, Zenker, formaldehyde alone or followed by dichromate or glutaraldehyde, freeze-substitution) were suitable for observation by fluorescence microscopy. Acetylation, deamination, and prolonged washing of stained sections with water, salt solution or ethanol strongly reduced eosin Y fluorescence, while it slightly increased after methylation. These results show that routine haematoxylin-eosin stained tissue sections can be routinely analysed by fluorescence microscopy. The emission of eosin Y allows easy and precise recognition of eosinophilic structures, which are poorly visible under bright field illumination.     

改良手工显微切割法获取特定细胞提取高质量RNA

探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100％无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要.     

Fixation techniques for fine needle aspiration biopsy smears prepared off site

OBJECTIVE: To identify a simple, cost-effective, reliable fixation method for fine needle aspiration biopsy (FNAB) yielding a specimen suitable for mail transport. STUDY DESIGN: Smears prepared from 59 FNABs of surgical specimens were fixed by continuous fixation in 95% ethanol, spray fixation, air drying, ethanol fixation for either 5 minutes or 4 hours followed by spray fixation, or fixation in 95% ethanol for either 30 minutes or 4 hours followed by air drying. Fixation was graded as unsatisfactory, suboptimal, average, good or excellent. RESULTS: Of smears continuously fixed in ethanol, 96.6% were graded as excellent. Of smears fixed in ethanol followed by spray fixation, 93.2% were excellent irrespective of fixation time; 64.4% of spray-fixed smears were excellent and 27.1% good. Of air dried smears, 93.2% were unsatisfactory or suboptimal; 83.0% of smears fixed in ethanol for 30 minutes and 74.6% of smears fixed for 4 hours prior to air drying were unsatisfactory or suboptimal. CONCLUSION: Fixation of smears in 95% ethanol followed by spray fixation produces excellent results, comparable to those with continuous fixation in ethanol. Spray fixation is generally good but not consistently excellent. Air drying or fixation in ethanol followed by air drying yields unsatisfactory or suboptimal results in most cases.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

Preparation of tissues for localization of sex hormones by autoradiography

Summary Tissues of rats given ³H-oestradiol were prepared for autoradiography according to methods commonly used in light and electron microscopy.By formalin fixation large amounts of radioactive material were lost, both in the fixative and during dehydration. Altogether 78.6±7.5 per cent was extracted from uterine tissue, while 49.0±4.6 per cent was lost from liver tissue removed 15 minutes after the injection. Significantly more radioactivity was lost in the fixative from liver tissue than from uterine. In the former fixation accounted for about 60 per cent of the loss, whereas in the latter it was responsible for about 25 per cent.Osmium tetroxide fixation was found to retain the radioactivity of liver and uterine tissue almost completely. However, large amounts were invariably extracted during dehydration. Although only 3.9±1.2 per cent of the radioactivity of uterine tissue diffused into the fixative, 72.8±12.4 per cent was extracted during ethanol dehydration. A heavy loss was also registered when dehydration and infiltration were carried out in glycol methacrylate.Glutaraldehyde perfusion and postfixation with osmium tetroxide retained almost completely the radioactivity of uterine and pituitary tissue. Nevertheless, nearly all of it was extracted during ethanol/propylene oxide dehydration and Epon embedding.The methods studied are not adequate for accurate autoradiographic localization of oestradiol.This work was supported by grants from The Norwegian Cancer Society and by Nordisk Insulinfond. The skilful assistance of Miss Helga Friedl and Mrs. Jane Larsen is gratefully acknowledged.     

Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cells.Electronic Supplementary Material Supplementary material is available for this article at     

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.     

Combined effects of formalin fixation and tissue processing on immunorecognition

Abstract

It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.     

The influence of fixatives and other variations in tissue processing on nuclear morphometric features

The influence on the area and numerical density of nuclei was investigated in 5-mm-thick slices of guinea pig liver for different fixatives and variations in tissue processing: delay in fixation, air drying, degree of acidity of 10% formalin (= 4% formaldehyde), Bouin and mercury-formalin fixatives, acetone and ethanol dehydration and understretching and overstretching of the paraffin-embedded sections. Air drying (either forced or as a result of delayed fixation), the type of fixative and the degree of acidity affected the nuclear area. Regarding the latter, nuclear area was approximately 25% lower for pH less than or equal to 3 as compared with pH greater than 5. In comparison with the standard tissue processing used, the nuclear density was higher after all of the variations studied (air drying, acetone dehydration and fixation). These findings indicate that nuclear area, in contrast to other tissue components, is relatively insensitive to variations in tissue processing. However, it is essential to regularly measure the pH of the fixative: deviations from pH = 7 should be carefully avoided in order to keep nuclear area variations as a result of tissue processing within acceptable limits.     

Studies of intermediary metabolism in germinating pea cotyledons. The pathway of ethanol metabolism and the role of the tricarboxylic acid cycle

1. The pathway of ethanol metabolism in cotyledons of 3-day-old pea seedlings has been examined by incubating tissue slices with [1-(14)C]ethanol and [2-(14)C]ethanol for periods up to 1hr. 2. Ethanol was rapidly incorporated into citrate and glutamate but relatively small amounts of (14)C were present in the evolved carbon dioxide even after 1hr. of ethanol metabolism. 3. Similar data were obtained from experiments in which [1,2-(14)C(2)]acetaldehyde and [(14)C]acetate were supplied. 4. The results are interpreted as indicating that ethanol is metabolized essentially via the reactions of the tricarboxylic acid cycle with a substantial drain of alpha-oxoglutarate to support the biosynthesis of glutamate. 5. It is concluded that oxaloacetate, required for the incorporation of ethanol into citrate, arises mainly from the transamination of aspartate and the fixation of carbon dioxide.     

Cytoskeletal elements in migrating and tentacle-integrated nematocytes of a marine hydrozoan

Summary— Actively migrating nematocytes of the marine polyp Stauridiosarsia producta are converted into completely immotile cells as soon as they become integrated in the ectodermal tissue of the tentacles. Immunocytochemical and electron microscopical methods revealed that cytoskeletal elements composed of actin, tubulin, centrin and a still unidentified protein are interwoven within the complete cell. While the organization of the sensory pole of the nematocyte, containing the cnidocil complex, the pseudovillar system and the distal half of a microtubular basket surrounding the nematocyst, is not affected by the transition from a motile to an immotile cell, the cytoskeletal elements in the basal portion of the cell are re-organized. Thus, the basolateral cytoplasm of migrating cells contains less organized microtubular arrays and bundles of about 10 nm-thick filaments. In the tentacle-integrated state, the 10-nm filaments are concentrated within a stalk-like foot which is stabilized by some rigid microtubular arrays derived from the microtubular basket. By elongation of the microtubular basket towards the cellular basis, the nematocyst becomes indirectly anchored at the mesoglea. As indicated by pharmacological treatments, the stiffness of the stalk depends on its microtubular content only.     

Notes on the application of microwaves in histopathology

Summary In this article two applications of microwaves in histopathology, microwave-stimulated staining of tissue sections and microwave-stimulated fixation of cryostat sections, are reviewed. For a good understanding of the influence of microwaves on physico-chemical processes like staining and fixation the relevant physics are included. Major advantages of microwave techniques are speed and/or improved quality. The cryostat-microwave technique appears to be well-suited for the demonstration of intermediate filament proteins: the sensitivity of monoclonal antibodies directed against keratins and vimentin can be substantially increased using the ethanol based fixative Kryofix.     

The effects of various fixatives on subsequent lectin binding to tissue sections

Summary The effects of ten fixation protocols on the subsequent binding of eight lectins to various mouse tissue sites have been systematically evaluated. The fixatives used were neutral and buffered formalin—saline, Bouin's fluid, 95% ethanol, Carnoy's fluid, calcium acetate—paraformaldehyde, and mercuric chloride both before and after removal of mercury pigment. These were compared with frozen sections of unfixed tissue and frozen sections post fixed in paraformaldehyde. Lectins used were PNA, DBA, SBA, BPA, UEA 1, GS I, GS II and MPA. Ethanol was found to be the superior fixative, closely followed by mercuric chloride. Paraformaldehyde was a poor fixative of both paraffin and frozen sections. It is recommended that, where a choice is possible, the fixation protocol appropriate to the particular lectin and tissue binding site is selected. Within certain limitations, formalin—saline proved an adequate fixative for the study of routine paraffin-processed tissue sections.     

Direct visualization of the microtubular cytoskeleton of ciliated protozoa with a fluorescent taxoid

Visualization of the infraciliature, which is an essential tool for the identification of ciliate species, has traditionally been obtained with silver proteinate methods. Since infraciliature is mainly composed of microtubules, we used the synthetic fluorescent taxoid FLUTAX as a method for ciliate identification. The main advantages of this method are the facility and rapidity of its application and the fact that no previous fixation and permeabilization processes are required. FLUTAX may also be used as a probe to follow morphogenetical changes in the microtubular cytoskeleton during the ciliate life cycle.