High Concentrations Of Glutaraldehyde Research Articles

Cyanophycin modifications for applications in tissue scaffolding

Cyanophycin (CGP) is a polypeptide consisting of amino acids—aspartic acid in the backbone and arginine in the side chain. Owing to its resemblance to cell adhesive motifs in the body, it can be considered suitable for use in biomedical applications as a novel component to facilitate cell attachment and tissue regeneration. Although it has vast potential applications, starting with nutrition, through drug delivery and tissue engineering to the production of value-added chemicals and biomaterials, CGP has not been brought to the industry yet. To develop scaffolds using CGP powder produced by bacteria, its properties (e.g., biocompatibility, morphology, biodegradability, and mechanical strength) should be tailored in terms of the requirements of the targeted tissue. Crosslinking commonly stands for a primary modification method for renovating biomaterial features to these extents. Herein, we aimed to crosslink CGP for the first time and present a comparative study of different methods of CGP crosslinking including chemical, physical, and enzymatic methods by utilizing glutaraldehyde (GTA), UV exposure, genipin, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), and monoamine oxidase (MAO). Crosslinking efficacy varied among the samples crosslinked via the different crosslinking methods. All crosslinked CGP were non-cytotoxic to L929 cells, except for the groups with higher GTA concentrations. We conclude that CGP is a promising candidate for scaffolding purposes to be used as part of a composite with other biomaterials to maintain the integrity of scaffolds. The initiative study demonstrated the unknown characteristics of crosslinked CGP, even though its feasibility for biomedical applications should be confirmed by further examinations.Key points• Cyanophycin was crosslinked by 5 different methods• Crosslinked cyanophycin is non-cytotoxic to L929 cells• Crosslinked cyanophycin is a promising new material for scaffolding purposesGraphical

Bacterial Resistance to Disinfectants in common office areas

Introduction. The use of different chemical agents for the attenuation, treatment and control of microorganisms has been increasing, the lack of control and knowledge of these products is generating a change in the genome in microorganisms, causing resistance to normal concentrations of biocides. Objective: To carry out a systematic review on bacterial resistance to disinfectants in common office areas. Methods: Systematic review of databases; Scielo, Elsevier, Pubmed and ACS Publications research, and secondary sources such as PAHO (Pan American Health Organization) and WHO (World Health Organization), among others, using terms such as; Bacterial resistance, disinfection, occupational or professional diseases and resistance to disinfectants. Results: Enterobacter sp.: resistant to Quaternary Ammonium (QAC), halogen-based disinfectants and 37% formaldehyde; Pseudomonas aeruginosa: 71% of isolates multiresistant to antibiotics, 43% reduced susceptibility to QAC, triclosan (TC) and Benzalkonium (BAC), and 24 isolates resistant to antimicrobial agents. M. massiliense BRA 100 susceptible to orthophthaldehyde (OPA), peracetic acid (PA), and high concentrations of glutaraldehyde. Clinical isolates of multiresistant strains to antibiotics such as: MRSA, Enterococcus sp. and Pseudomonas aeruginosa, 52% and 38% strains were resistant to quaternary ammonium and phenol compounds, respectively. Conclusions: The presence of resistant microorganisms in common places such as; floors, light switches, door handles, desks and chairs, among others, enunciates a public health problem that must begin to be addressed, changing the methodologies used for disinfection, and other control and prevention measures.

Effect of microtubule immobilization by glutaraldehyde on kinesin-driven cargo transport

The glutaraldehyde fixation method for fixing tissues is attractive for its ease of use and straightforward surface chemistry. We investigated the effect of glutaraldehyde-induced microtubule immobilization on kinesin-driven cargo transport along microtubules and found that at low glutaraldehyde concentrations, the microtubule–kinesin interaction remains unperturbed. Such findings may facilitate the application of the glutaraldehyde fixation method for many in vitro studies aiming to build nanodevices powered by the microtubule–motor protein interaction. The graphical abstract shows that the kinesin-driven quantum dot transport along microtubule immobilized on a substrate using glutaraldehyde concentration ≤0.10% (v/v) remain unaltered, whereas, at higher glutaraldehyde concentration, >0.10% (v/v), the quantum dot transportation is slowed down.

Chemical cross-linking: A feasible approach to prolong doxylamine/pyridoxine release from spray-dried chitosan microspheres

Spray-dried chitosan microparticles have been widely exploited as vehicles for mucosal drug delivery. Despite their advantages as pharmaceutical formulations, one of the major challenges is achieving sustained drug release, which would diminish toxicity and dosage frequency. The aim of this study was to formulate mucoadhesive glutaraldehyde cross-linked chitosan microparticles loaded with doxylamine succinate and pyridoxine hydrochloride as potential nasal drug delivery systems with sustained release. Microparticle models were formulated via spray-drying technique, using glutaraldehyde in different concentrations (0.1–1.0 mg/mL) as a cross-linking agent for chitosan. The obtained particles were with spherical shape, smooth surface and median diameter of 4 μm. The drug entrapment efficiency was high (80.47%–94.25%), indicating a tendency to decrease at higher glutaraldehyde concentrations. FTIR data demonstrated that there were no chemical interactions between glutaraldehyde and the drugs. The in vitro studies showed that the cross-linking process substantially limited particles swelling. The cross-linked particles exhibited sustained drug release characteristics at pH 6.8 over a period of 5 h with an initial burst-effect in the first 30 min. Drug release followed Korsmeyer-Peppas kinetics. Although a decrease of the particles mucoadhesive properties was observed after modification, all cross-linked formulations demonstrated high in vitro adsorption of mucin. The proposed models of mucoadhesive microsphere with sustained drug release are a perspective ground for further development of a novel delivery system for nasal administration of doxylamine and pyridoxine.

Immobilization of β-1,3-1,4-glucanase from Bacillus sp. on porous silica for production of β-glucooligosaccharides.

(1,3)(1,4)-β-d-glucan has been determined to have various beneficial effects due to its unique structure. β-glucooligosaccharides (β-GOS), which are hydrolysates of barley (1,3)(1,4)-β-d-glucan, provide a useful prebiotic material for selective growth of probiotic bacteria. In this study, recombinant β-1,3-1,4-glucanase (Bg1314) from Bacillus sp. SJ-10 (KCCM 90078) was immobilized on porous silica using glutaraldehyde as a crosslinking reagent to achieve efficient production of β-GOS. We investigated the effects of factors such as the amounts of enzyme and glutaraldehyde, reaction temperature, and pH on catalytic activity. Enzyme activity decreased sharply at high concentrations of glutaraldehyde, likely due to the reaction of glutaraldehyde with lysine residues at the catalytic site of Bg1314, because lysine-substituted Bg1314 retained its activity under the same conditions. Immobilized Bg1314 protein (ImBg1314) was stable over a wide range of pH and could be stored long term at 4 °C. The optimal conditions of ImBg1314 were similar to those of Bg1314. However, the optimal temperature of ImBg1314 differed from that of Bg1314. The products were β-GOS composed of 3-O-β-cellobiosyl-d-glucose and 3-O-β-cellotriosyl-d-glucose. After ImBg1314 was reused for 10 cycles, it retained 42% of its initial catalytic activity. This study showed that the Imbg1314 applied economical production of β-GOS.

Evaluation of biodegradation and biocompatibility of collagen/chitosan/alkaline phosphatase biopolymeric membranes

The aim of this study was to develop a new variant of membranes based on collagen (COL), chitosan (CHI) and alkaline phosphatase (ALP) immobilized and cross-linking with glutaraldehyde (GA) at different concentrations. The biodegradation in the presence of collagenase was investigated. Biocompatibility was evaluated by MTT assay using a mouse fibroblast cell culture type NCTC (clone 929). Non-cross-linked samples were biocompatible and membranes cross-linked with low concentrations of GA (0.04, 0.08%) were also biocompatible. However, high concentrations of GA lead to a decreased biocompatibility. The adsorption behaviour of Ca2+ ions to all membranes were evaluated using the Freundlich isotherms. Haemolytic studies were performed in order to consider their applications in biomineralization process. By the addition of collagen and ALP to chitosan, the haemolytic index decreases, the COL–CHI–ALP membrane being in the non-haemolytic domain, while the COL–CHI–ALP–GA membrane has a haemolytic index greater than 2, and is slightly haemolytic.

Architectural and mechanical cues direct mesenchymal stem cell interactions with crosslinked gelatin scaffolds.

Naturally derived biomaterials have emerged as modulators of cell function and tissue substitutes. Here, we developed crosslinked glutaraldehyde (GTA) scaffolds for the expansion and differentiation of mesenchymal stem cells (MSCs). The mechanical and architectural properties of the scaffolds were altered by varying the concentration of gelatin and GTA. Higher GTA concentrations were associated with an increase in more confined pores and osteogenic differentiation. In addition, myogenic potential varied with crosslinking degree, although bulk mechanical properties were unaltered. Correlation analysis revealed that ALP activity of differentiated MSCs on higher gelatin concentration scaffolds was dependent on traditional effectors, including environment elasticity and spread area. In contrast, the differentiation capacity of cells cultured on lower gelatin concentration scaffolds did not correlate with these factors, instead it was dependent on the hydrated pore structure. These results suggest that scaffold composition can determine what factors direct differentiation and may have critical implications for biomaterial design.

Immunohistochemical staining for neurotransmitter‐related antigens in nerves fixed with high concentrations of glutaraldehyde (726.7)

Immunohistochemistry (IHC) demonstrates specific antigens within tissues or cells and is usually done on formaldehyde‐fixed tissues. However, for a detailed quantification of axons within nerve bundles, glutaraldehyde fixation is mandatory. We aimed to develop an IHC technique for nerves fixed with high concentrations of glutaraldehyde. Phrenic nerves from 8 male Wistar rats were removed, fixed in phosphate‐buffered 2.5% glutaraldehyde and cryoprotected in 30% sucrose. Slide‐mounted cryostat sections (16 µm thick) were incubated for 24 hr in goat polyclonal anti‐choline acetyltransferase (ChAT; 1:1,000) or rabbit polyclonal anti‐substance P (SP; 1:10,000) then transferred to biotinylated donkey anti‐goat or donkey anti‐rabbit immunoglobulin (1:500; 24 hr) before immersion in Extravidin‐HRP (1:1.500; 24 hr). Immunoreactivity in axons was revealed with a nickel‐intensified diaminobenzidine reaction. This method reproducibly revealed individual axons that contained immunoreactivity for ChAT or SP within the phrenic nerve. These results show that peptides and large proteins can be detected by IHC in nerves that have been immersion‐fixed in a high concentration of glutaraldehyde. This method is reliable for identifying neurotransmitter‐related antigens and will allow quantification of axons with specific physiological roles, which can be inferred from their neurochemical phenotypes.Grant Funding Source: FAPESP, CAPES, CNPq and FAEPA

Electron microscopic identification of hydrogen peroxide detected in fixed human polymorphonuclear leukocytes during phagocytosis.

Polymorphonuclear leukocytes (PMNs) engaged in phagocytosis produce reactive oxygen species (ROS), such as those that occur in an activated NADPH oxidase reaction, to eliminate ingested microorganisms. The translocation of NADPH oxidase components to produce antimicrobial free radicals from the vesicles to the phagosomes may be important. Hydrogen peroxide (H2O2) derived from O2- has been observed by electron microscopy using a cerium method. However, 2'-7'-dichlorofluorescin diacetate can also detect H2O2 through fluorescence. The main objective of the present study was to measure the H2O2-dependent fluorescence of PMNs after opsonized zymosan A (OPZ) phagocytosis using a microplate reader under different fixation conditions, including 0.5, 1, and 10% glutaraldehyde (GA) individually for 1, 5, 10, or 30 min. An additional objective was to visualize, through the use of electron microscopic cytochemistry, the process of H2O2 generation in OPZ phagocytic fixed PMNs. The fixed PMNs showed that the largest fluorescent value was produced by a concentration of 0.5% GA for all fixation times. This suggested that the fixation of PMNs with a high concentration of GA inhibited phagocytosis and produced ROS. In the fixed PMNs, electron microscopic results showed that after 1 min of mixing, some PMNs attached to particles and exhibited mild deposits in their secretory vesicles. When PMNs engulfed particles, free radical-producing vesicles had enhanced reaction deposits 10 min later and fused to the phagosomal membrane, releasing numerous free radicals into the lumen. Time-dependent H2O2 production was enhanced in the secretory vesicles, some of which were fused exactly to the phagosome membranes.

Start-up shape dynamics of red blood cells in microcapillary flow

Red blood cell (RBC) deformability plays a key role in oxygen exchange between blood and tissues in microcirculation by allowing RBCs to flow in vessels of diameter even smaller than cell size. Hence, RBC flow in microcapillaries has been widely studied in vitro, mostly under steady-state conditions. Here, we provide the first quantitative investigation of the transient behavior of RBC shape in confined Poiseuille flow in vitro. Our approach is based on high-speed video microscopy imaging of RBCs flowing in silica microcapillaries and quantitative data processing by image analysis techniques. In start-up flow, RBCs undergo a complex transition from the biconcave shape to a parachute-like configuration through membrane folding and cytoplasm reorganization. The time scale of this transient process is independent on the applied pressure drop and the measured value for healthy cells (around 0.1 s) is in agreement with previous micropipette data from the literature. Glutaraldehyde (GA)-hardened RBCs exhibit a faster shape evolution at higher GA concentration, thus showing that the corresponding time scale becomes shorter at increasing cytoskeleton elasticity. Our results provide a novel microfluidics methodology to measure the RBC characteristic time which is a potential diagnostic parameter of altered cell deformability.

Mycobacterium massiliense BRA100 strain recovered from postsurgical infections: resistance to high concentrations of glutaraldehyde and alternative solutions for high level disinfection

To evaluate the minimum inhibitory concentration (MIC) of GTA against these microorganisms and alternative disinfectants for high-level disinfection (HLD). Reference mycobacteria and clinical M. massiliense strains were included in this study. Active cultures were submitted to susceptibility qualitative tests with GTA dilutions (ranging from 1.5% to 8%), and commercial orthophthaldehyde (OPA) and peracetic acid (PA)-based solutions, during the period of exposure as recommended by National Agency of Sanitary Surveillance for HLD. All reference and M. massiliense non-BRA100 strains, recovered from sputum, were susceptible to any GTA concentration, OPA and PA solutions. M. massiliense BRA100 strains presented MIC of 8% GTA and were susceptible to OPA and PA. M. massiliense BRA100 strain is resistant to high GTA concentrations (up to 7%), which proves that this product is non-effective against specific rapidly growing mycobacteria and should be substituted by OPA or PA-based solutions for HLD.

Polyaniline nanoparticles with controlled sizes using a cross-linked carboxymethyl chitin template

Polyaniline (PANI) nanoparticles were chemically synthesized in the presence of a cross-linked carboxymethyl chitin (CM-chitin) acting as a template. The reaction was performed under acidic conditions and the template was removed after the polymerization of aniline was completed. The morphology of the synthesized PANI was globular with a diameter in the nanometer range. The degree of cross-linking of the CM-chitin played an important role in determining the size of the obtained PANI nanoparticles, which decreased from approximately 392 to 160 nm with increase in concentration of the cross-linking agent, glutaraldehyde, from 0 to 9 μmol, respectively. At a higher glutaraldehyde concentration (18 μmol), an aggregated PANI network was observed due to the incomplete removal of the more highly cross-linked CM-chitin. Molecular characterization (including UV-Visible, FTIR, TGA, and XRD techniques) revealed that the structure of the synthesized PANI nanoparticles is identical to that of conventional PANI. A mechanism is proposed for the formation of PANI nanoparticles in the presence of the cross-linked CM-chitin template.

A new crosslinked protein fiber from gliadin and the effect of crosslinking parameters on its mechanical properties and water stability

AbstractBACKGROUND: Although several cereal proteins have been used to develop fibers and films, it has not been possible to obtain protein materials with good mechanical properties and water stability, even after crosslinking. Previously, high concentrations of glutaraldehyde were used to improve the mechanical properties of protein fibers but the effect of crosslinking conditions on the properties of the crosslinked materials has not been studied in detail.RESULTS: Low concentrations of glutaraldehyde can be used to improve the mechanical properties and water stability of gliadin fibers. Quantitative relationships that can predict the breaking tenacity of the fibers at various crosslinking conditions are developed. Glutaraldehyde crosslinking is more resistant to hydrolysis in neutral pH than under acidic conditions in terms of increasing and retaining the breaking tenacity. The crosslinked fibers show improved resistance to hydrolysis over poly(lactic acid) fibers in aqueous dispersions at pH = 4 and 7 at 50 and 90 °C, respectively.CONCLUSIONS: This study shows that low concentrations of glutaraldehyde can impart excellent mechanical properties to gliadin fibers. The quantitative relationships developed can be used to select the crosslinking conditions such low glutaraldehyde concentration and high temperature or vice versa to obtain the desired improvement in mechanical properties or water stability. Copyright © 2008 Society of Chemical Industry

Monodisperse Gelatin Microspheres as a Drug Delivery Vehicle: Release Profile and Effect of Crosslinking Density

Uniform gelatin microspheres (GMS) of a wet size of 100 microm in diameter were fabricated by the electric field assisted precision particle fabrication (E-PPF) method and crosslinked with different glutaraldehyde (GA) concentrations to study the effect of the crosslinking density on drug release. The drug release profiles of the crosslinked GMS were studied along with the intraparticle drug distribution and the particle degradation characteristics. Due to the concentration gradient of GA along the diffusion path into the GMS, the crosslinking density is higher on the GMS surface, making it less susceptible to degradation. As a result, the GMS with higher GA concentrations (0.375-0.875%) exhibited a highly resistant surface toward enzymatic degradation. On the other hand, the amount of drug complexation at the surface decreases as the GA concentration increases, which can be attributed to the lowered basicity of gelatin caused by the increased crosslinking density. These factors collectively affect the drug release kinetics and give rise to similar release profiles for GMS above a GA concentration of 0.375%.

Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder

Addition of bovine serum albumin (BSA) as a proteic feeder facilitates obtaining cross-linked enzyme aggregates (CLEAs) in cases where the protein concentration in the enzyme preparation is low and/or the enzyme activity is vulnerable to the high concentration of glutaraldehyde required to obtain aggregates. CLEAs of Pseudomonas cepacia lipase and penicillin acylase were prepared. CLEA of lipase prepared in the presence of BSA retained 100% activity whereas CLEA prepared without BSA retained only 0.4% activity of the starting enzyme preparation. Lipase CLEA showed 12-fold increase in activity over free enzyme powder when the CLEA was used in transesterification of tributyrin. For the transesterification of Jatropha oil, while free enzyme powder required 8 h and 50 mg lipase to obtain 77% conversion, CLEA required only 6 h and 6.25 mg lipase to obtain 90% conversion. In the case of penicillin acylase, 86% activity could be retained in CLEA prepared with BSA whereas CLEA made without BSA retained only 50% activity. CLEA prepared without BSA lost 20% activity after 8 h at 45 °C whereas CLEA with BSA retained full activity. CLEA prepared with BSA showed V max/ K m of 36.3 min −1 whereas CLEA prepared without BSA had V max/ K m of 17.4 min −1 only. Scanning electron microscopy analysis showed that CLEAs prepared in the presence of BSA were less amorphous and closer in morphology to CLEAs of other enzymes described in the literature.

Inhibition of diethyl ether degradation in Rhodococcus sp. strain DEE5151 by glutaraldehyde and ethyl vinyl ether

Alkyl ether-degrading Rhodococcus sp. strain DEE5151, isolated from activated sewage sludge, has an activity for the oxidation of a variety of alkyl ethers, aralkyl ethers and dibenzyl ether. The whole cell activity for diethyl ether oxidation was effectively inhibited by 2,3-dihydrofurane, ethyl vinyl ether and glutaraldehyde. Glutaraldehyde of less than 30 μM inhibited the activity by a competitive manner with the inhibition constant, K I of 7.07 ± 1.36 μM. The inhibition type became mixed at higher glutaraldehyde concentrations >30 μM, probably due to the inactivation of the cell activity by the Schiff-base formation. Structurally analogous ethyl vinyl ether inhibited the diethyl ether oxidation activity in a mixed manner with decreasing the apparent maximum oxidation rate, v max app , and increasing the apparent Michaelis–Menten constant, K M app . The mixed type inhibition by ethyl vinyl ether seemed to be introduced not only by the structure similarity with diethyl ether, but also by the reactivity of the vinyl ether with cellular components in the whole cell system.

Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering

Porous scaffolds for skin tissue engineering were fabricated by freeze-drying the mixture of collagen and chitosan solutions. Glutaraldehyde (GA) was used to treat the scaffolds to improve their biostability. Confocal laser scanning microscopy observation confirmed the even distribution of these two constituent materials in the scaffold. The GA concentrations have a slight effect on the cross-section morphology and the swelling ratios of the cross-linked scaffolds. The collagenase digestion test proved that the presence of chitosan can obviously improve the biostability of the collagen/chitosan scaffold under the GA treatment, where chitosan might function as a cross-linking bridge. A detail investigation found that a steady increase of the biostability of the collagen/chitosan scaffold was achieved when GA concentration was lower than 0.1%, then was less influenced at a still higher GA concentration up to 0.25%. In vitro culture of human dermal fibroblasts proved that the GA-treated scaffold could retain the original good cytocompatibility of collagen to effectively accelerate cell infiltration and proliferation. In vivo animal tests further revealed that the scaffold could sufficiently support and accelerate the fibroblasts infiltration from the surrounding tissue. Immunohistochemistry analysis of the scaffold embedded for 28 days indicated that the biodegradation of the 0.25% GA-treated scaffold is a long-term process. All these results suggest that collagen/chitosan scaffold cross-linked by GA is a potential candidate for dermal equivalent with enhanced biostability and good biocompatibility.

Disinfection of striped trumpeter (Latris lineata) eggs with glutaraldehyde

This study assessed the effect of immersing striped trumpeter eggs in 0 (control), 200, 400, 800, 1600 or 3200 ppm glutaraldehyde for 10 minutes, two days before hatching. High concentrations of glutaraldehyde (1600 and 3200 ppm) resulted in no eggs hatching and only 1% of eggs hatched after treatment with 800 ppm glutaraldehyde. Hatching success of eggs treated with 0, 200 or 400 ppm glutaraldehyde did not differ (77 ± 6%, n = 3). However, only 2% of larvae from the control treatment survived to day 5 post-hatching, compared to 45 and 69%, respectively of the larvae from the 200 and 400 ppm glutaraldehyde treatment. By day 9 post-hatching, larvae from the 400 ppm glutaraldehyde treatment had significantly higher survival (59%) than larvae from the 200 ppm glutaraldehyde treatment (28%). Thiosulphate citrate bile salt sucrose agar (TCBS) medium confirmed the presence of bacteria within the seawater medium, on all control eggs and on 83% of eggs disinfected with 200 ppm glutaraldehyde, but no bacterial colonies formed on eggs treated with 400, 800, 1600 or 3200 ppm glutaraldehyde. This study found highest survival of striped trumpeter larvae from eggs disinfected with 400 ppm glutaraldehyde and suggests that increased survival was a result of reduced bacterial loading.

LR white post-embedding colloidal gold method to immunostain MBP, P0, NF and S100 in glutaraldehyde fixed peripheral nerve tissue.

A variety of immunocytochemical techniques are now widely used for the electron and light microscopic examination of biological samples. They are employed routinely for investigating the role of certain proteins in nervous tissue. Immunoelectron microscopic studies require the tissue to be fixed and embedded in a solid support, which may disrupt cellular structures and destroy crucial antigens. A technique of post-embedding with LR white resin has been developed, and it has been shown that certain antigens tolerate fixation with glutaraldehyde. In this study, we optimized a previous post-embedding method using low-water-miscible low-temperature embedding resin (LR white) to immunostain MBP, P0, NF and S100 proteins in peripheral nerves fixed with a relatively high concentration of glutaraldehyde found to be compatible with the morphology of normally compacted nerve fibers from humans and adult animals. The main difference in the procedures described here from previous ones is the elimination of vibratome sectioning, rendering this immunostaining technique more accessible to neuropathological laboratories using standard equipment for the ultrastructural study of peripheral nerves. It may prove of value for localization and quantification of these proteins in normal and pathological conditions.

Phalloidin-eosin followed by photo-oxidation: a novel method for localizing F-actin at the light and electron microscopic levels.

We describe a novel high-resolution method to detect F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidation of diaminobenzidine. This method possesses several key advantages over antibody-based labeling and structural methods. First, phalloidin binding to F-actin can tolerate relatively high concentrations of glutaraldehyde (up to 1%) in the primary fixative, resulting in good ultrastructural preservation. Second, because both eosin and phalloidin are relatively small molecules, considerable penetration of reagents into aldehyde-fixed tissue was obtained without any permeabilization steps, allowing 3D reconstructions at the electron microscopic level. By employing a secondary fixation with tannic acid combined with low pH osmication, conditions known to stabilize actin filaments during preparation for electron microscopy, we were able to visualize individual actin filaments in some structures. Finally, we show that fluorescent phalloidin can be directly injected into neurons to label actin-rich structures such as dendritic spines. These results suggest that the fluorescent phalloidin is an excellent tool for the study of actin networks at high resolution.