Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.565
Filtrar
1.
Mol Ecol Resour ; 23(1): 92-105, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35932285

RESUMO

DNA metabarcoding is routinely used for biodiversity assessment, in particular targeting highly diverse groups for which limited taxonomic expertise is available. Various protocols are currently in use, although standardization is key to its application in large-scale monitoring. DNA metabarcoding of arthropod bulk samples can be conducted either destructively from sample tissue, or nondestructively from sample fixative or lysis buffer. Nondestructive methods are highly desirable for the preservation of sample integrity but have yet to be experimentally evaluated in detail. Here, we compare diversity estimates from 14 size-sorted Malaise trap samples processed consecutively with three nondestructive approaches (one using fixative ethanol and two using lysis buffers) and one destructive approach (using homogenized tissue). Extraction from commercial lysis buffer yielded comparable species richness and high overlap in species composition to the ground tissue extracts. A significantly divergent community was detected from preservative ethanol-based DNA extraction. No consistent trend in species richness was found with increasing incubation time in lysis buffer. These results indicate that nondestructive DNA extraction from incubation in lysis buffer could provide a comparable alternative to destructive approaches with the added advantage of preserving the specimens for postmetabarcoding taxonomic work but at a higher cost per sample.


Assuntos
Artrópodes , Animais , Artrópodes/genética , Código de Barras de DNA Taxonômico/métodos , Fixadores , Biodiversidade , DNA/genética , Etanol
2.
Methods Mol Biol ; 2561: 63-85, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36399265

RESUMO

In this protocol, we describe the specific steps required to prepare human postmortem brain samples for ultrastructural microglial analysis. A detailed procedure is provided to improve the ultrastructural quality of the samples, using aldehyde fixatives followed by immunoperoxidase staining of allograft inflammatory factor 1 (AIF1, also known as IBA1), a marker of myeloid cells, and cluster of differentiation 68 (CD68), a marker of phagolysosomal activity. Additionally, we describe an osmium-thiocarbohydrazide-osmium (OTO) post-fixation method that preserves and increases the contrast of cellular membranes in human postmortem brain samples, as well as the steps necessary to acquire scanning electron microscopy (SEM) images of microglial cell bodies. In the last section, we cover the quantitative analysis of various microglial cytoplasmic organelles and their interactions with other parenchymal elements.


Assuntos
Encéfalo , Microglia , Humanos , Microglia/ultraestrutura , Microscopia Eletrônica de Varredura , Autopsia , Fixadores
3.
Methods Mol Biol ; 2566: 159-171, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152250

RESUMO

Potassium permanganate solution has been used both as a fixative and as a staining for ultrathin sections at transmission electron microscopy, due to its ability to provide good contrast of different tissue components. Subsequently, it has been forgotten due to disadvantages such as conspicuous formation of precipitates and fragility of the tissue sections treated with this dye when placed under the electron beam. Here we demonstrate that the observed granularity of the sections is not related to the formation of non-specific precipitates, but rather to basic proteins such as chromatin proteins closely associated with DNA and ribosomal particles which are intensely stained. This results in a marked contrast of the nuclei, in particular of the heterochromatin areas, the granular component of the nucleoli, and the rough endoplasmic reticulum, that are rich in these protein complexes. We also show how the embedding in LR white acrylic resin can preserve a good morphology and be less sensitive to the treatment with potassium permanganate than the epoxy resin sections, also allowing to perform immunocytochemistry. The fragility of the epoxy resin sections can be partially improved by using formvar-coated grids.


Assuntos
Corantes , Permanganato de Potássio , Resinas Acrílicas , DNA , Resinas Epóxi , Fixadores , Heterocromatina , Microscopia Eletrônica de Transmissão
4.
Acta Histochem ; 124(8): 151962, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36228481

RESUMO

BACKGROUND AND PURPOSE: In order to acquire the best method that can simultaneously maximize tissue morphology and staining quality, we compared the effect of different fixative and decalcifying solutions on the quality of rabbit and rat bone histology. METHOD: Fifty-four rat hemimaxillae and 54 rabbit quarter-parietal bones were allocated into 3 fixation groups (formalin, 10 %sodium-phosphate-buffered-formalin and 10 %calcium-phosphate-buffered-formalin). Each fixative was divided into 6 groups and decalcified with 5 % and 10 % nitric acid (NA), 5 % and 10 % formic acid (FA), Gooding-Stewart liquid (GSL) and EDTA. Slide quality was evaluated on hematoxylin/eosin slides by 3 observers and mean-scores for total-cell-characteristics (TCC) and total-tissue-characteristics (TTC) were statistically analyzed. RESULT: Significant differences in decalcification-time were observed in different combinations of decalcifiers and fixatives in both animals. In rats, TCC was better preserved when using 10 %NA/calcium-phosphate-buffered-formalin compared to 10 %NA/sodium-phosphate-buffered-formalin (P = 0.03). GSL/sodium-phosphate-buffered-formalin performed better than both other fixatives (P < 0.001). TCC differed among the decalcifiers in each of the fixatives. In rabbits, there were differences in TCC among the decalcifiers when formalin (P = 0.001) and sodium-phosphate-buffered-formalin (P = 0.01) were used. TTC only showed significant difference when 10 %FA was used in rats (P = 0.044), with formalin performing better than sodium-phosphate-buffered-formalin (P = 0.01). CONCLUSION: Based on our results, if time is an issue, 10 %NA/calcium-phosphate-buffered-formalin could provide good cellular quality and if time is not a consideration, FA (5 % or 10 %) with sodium-phosphate-buffered-formalin followed by EDTA with formalin, would have the best performance. In rabbits, GSL provides the fastest results, regardless of the fixative and FA/sodium-phosphate-buffered-formalin gives the best cellular quality.


Assuntos
Cálcio , Formaldeído , Coelhos , Ratos , Animais , Fixadores/farmacologia , Ácido Edético , Fosfatos , Sódio , Fixação de Tecidos/métodos
5.
Harmful Algae ; 118: 102311, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36195425

RESUMO

Numerous products and techniques are used to combat harmful cyanobacterial blooms in lakes. In this study, we tested nine products, the phosphate binders Phoslock® and Aqual-PTM, the coagulant chitosan, the phosphorus binder and coagulant aluminum salts (aluminum sulphate and sodium aluminate), the copper-based algicides SeClear, Captain® XTR and CuSO4·5H2O, the antibiotic Streptomycin and the oxidant hydrogen peroxide (H2O2) on their efficiency to manage the cyanobacterium Microcystis aeruginosa (M. aeruginosa). To this end, 7 days of laboratory experiments were conducted and effects were determined on chlorophyll-a, photosystem II efficiency (PSII), soluble reactive phosphorus (SRP) and intracellular and extracellular microcystin (MC) concentrations. The algicides, chitosan and H2O2 were the most powerful in reducing cyanobacteria biomass. Biomass reductions compared to the controls yielded: Chitosan (99.8%) > Hydrogen peroxide (99.6%) > Captain XTR (98.2%) > SeClear (98.1%) > CuSO4·5H2O (97.8%) > Streptomycin (86.6%) > Phoslock® (42.6%) > Aqual-PTM (28.4%) > alum (5.5%). Compounds that caused the largest reductions in biomass also strongly lowered photosystem II efficiency, while the other compounds (Phoslock®, Aqual-PTM, aluminum salts) had no effect on PSII, but strongly reduced SRP. Intracellular MC concentration followed the biomass patterns, extracellular MC was generally lower at higher doses of algicides, chitosan and H2O2 after one week. Recovery of PSII was observed in most algicides and chitosan, but not at the highest doses of SeClear and in all streptomycin treatments. Our results revealed that M. aeruginosa can be killed rapidly using several compounds, that in some treatments already signs of recovery occurred within one week. P fixatives are efficient in reducing SRP, and thus acting via resource suppression, which potentially may provide an addition to fast-acting algicides that kill most of the cells, but allow rapid regrowth as sufficient nutrients remain.


Assuntos
Quitosana , Cianobactérias , Herbicidas , Microcystis , Alumínio/farmacologia , Antibacterianos/farmacologia , Quitosana/farmacologia , Clorofila , Cobre/farmacologia , Fixadores/farmacologia , Herbicidas/farmacologia , Peróxido de Hidrogênio , Microcistinas/farmacologia , Oxidantes/farmacologia , Fosfatos , Fósforo/farmacologia , Complexo de Proteína do Fotossistema II , Sais/farmacologia , Estreptomicina/farmacologia , Sulfatos/farmacologia
6.
Medicine (Baltimore) ; 101(36): e30449, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36086724

RESUMO

EUS-guided fine needle aspiration cytology (FNA) is the gold standard of evaluation of solid pancreatic lesions. However, accuracy is generally low. The aim of this study was to compare the diagnostic yield of conventional cytology (CC) with liquid-based cytological analysis using an ethanol based fixative system (LBC) without onsite cytopathological assessment. We performed a retrospective evaluation in patients referred to the Department of Interdisciplinary Endoscopy at Jena University Hospital for FNA of pancreatic masses between 2008 and 2015. LBC preservation of specimen was introduced in April 2011. Gold standard was defined as a surgically obtained histology or a patient follow-up of at least 1 year for diagnosis or exclusion of malignancy. 172 patients were included into the final analysis. Mean age was 64.8 years (SD 12.4 years), 105 patients were male. 107 lesions were malignant, while 65 lesions were benign. 89 specimens were evaluated by CC, whereas 83 specimens were processed by LBC. Liquid-based cytology performed significantly better than conventional cytology in terms of sensitivity (87.8% vs 67.2% (P = .021)), specificity (100% vs 87.1% (P = .047)) negative predictive value (NPV) (85% vs 58.7% (P = .009)) and accuracy (92.8% vs 74.2% (P = .001)). We observed no learning curve after implementation of LBC Liquid based cytology is a simple and inexpensive technique that helps improving sensitivity, specificity, NPV and accuracy over conventional cytology in fine needle aspirates from patients with pancreatic lesions. Therefore, this real-world evidence shows, that EUS-FNA specimen processing should be performed using LBC to achieve best possible results.


Assuntos
Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico , Neoplasias Pancreáticas , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/métodos , Etanol , Feminino , Fixadores , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Estudos Retrospectivos
7.
Acta Histochem ; 124(7): 151952, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36099745

RESUMO

Immunohistochemistry (IHC) is a powerful biochemical technique that uses antibodies to specifically label and visualize proteins of interests within biological samples. However, fluid-preserved specimens within natural history collection often use fixatives and protocols that induce high background signal (autofluorescence), which hampers IHC as it produces low signal-to-noise ratio. Here, we explored techniques to reduce autofluorescence using sodium borohydride (SBH), citrate buffer, and their combination on fish tissue preserved with paraformaldehyde, formaldehyde, ethanol, and glutaraldehyde. We found SBH was the most effective quenching technique, and applied this pretreatment to the gill or skin of 10 different archival fishes - including specimens that had been preserved in formaldehyde or ethanol for up to 65 and 37 years, respectively. The enzyme Na+/K+-ATPase (NKA) was successfully immunostained and imaged using confocal fluorescence microscopy, allowing for the identification and characterization of NKA-rich ionocytes essential for fish ionic and acid-base homeostasis. Altogether, our SBH-based method facilitates the use of IHC on archival samples, and unlocks the historical record on fish biological responses to environmental factors (such as climate change) using specimens from natural history collections that were preserved decades to centuries ago.


Assuntos
Formaldeído , Museus , Adenosina Trifosfatases , Animais , Boroidretos , Citratos , Etanol , Peixes , Fixadores , Formaldeído/química , Glutaral
8.
J Histotechnol ; 45(4): 172-181, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36111534

RESUMO

Investigating the function of delicate mammalian eyes often requires chemical fixation, histological sectioning, immunohistochemistry (IHC) and in situ hybridization (ISH). One of the long-standing challenges in the ocular histology field is the limited success of maintaining intact morphology via cryo- or paraffin procedures. Although our latest protocol significantly improved the morphology of mouse eyeball sections, the window technique is time-consuming and requires extensive practice to avoid damage while making windows. In this study, we present a novel glyoxal fixative that is suitable for a freeze-substitution approach to improve both morphology and molecular target preservation of mouse eyes. The method prevents morphology distortion in all tested eyeballs. Therefore, it suits a variety of research needs from morphological examination to investigation of single-molecule RNA expression, using hematoxylin and eosin (H&E) stain, IHC, and ISH assays on either frozen (cryo) or paraffin-infiltrated tissue sections. In addition, this method can be easily performed in many histology laboratories.


Assuntos
Glioxal , Parafina , Animais , Camundongos , Fixadores/farmacologia , Glioxal/farmacologia , Solventes , Hibridização In Situ , Mamíferos
9.
Andrology ; 10(8): 1660-1672, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36082398

RESUMO

BACKGROUND: The unique anatomy of the male reproductive organ reflects its complex function from sperm maturation to their storage for months until emission. Since light microscopy in two dimensions (2d) cannot sufficiently demonstrate its complex morphology, a comprehensive visualization is required to identify pathologic alterations in its entire anatomical context. OBJECTIVES: Aim of this study was to use three-dimensional (3d) light sheet fluorescence microscopy (LSFM) to visualize entire murine testes in 3d, label-free and at subcellular resolution, and to assign local autofluorescence to testicular and deferent structures. MATERIALS AND METHODS: Murine testes were fixed with four different fixatives and subsequently cleared with benzoic acid/benzyl benzoate. Hereafter, complete murine testes were scanned with LSFM with different fluorescence filter sets and subsequently embedded in paraffin for further conventional planar histology. RESULTS: Autofluorescence signals of the murine reproductive organ allowed the unambiguous identification of the testicular anatomy from the seminiferous tubules to the vas deferens with their specific stratification independent of the used fixative. Blood vessels were visualized from the pampiniform plexus to the small capillaries of single tubules. Moreover, due to the specific intrinsic fluorescence properties of the efferent ducts and the epididymis, luminal caliber, the epithelial stratification and retronuclear cytoplasmic inclusions gave a unique insight into the interface of both morphological structures. Subsequent 2d histology confirmed the identified morphological structures. DISCUSSION: LSFM analysis of the murine reproductive organ allows due to its intrinsic fluorescence a simple, label-free 3d assessment of its entire duct morphology, the epithelial composition, and the associated blood supply in its anatomical relation. CONCLUSION: LSFM provides the technical basis for comprehensive analyses of pathologically altered murine testes in its entirety by depicting specific autofluorescence. Thereby it facilitates mouse studies of testicular disease or their drug-related alterations in more detail potentially for clinical translation assessing human testicular biopsies.


Assuntos
Parafina , Sêmen , Animais , Ácido Benzoico , Epididimo/patologia , Fixadores , Humanos , Masculino , Camundongos , Microscopia de Fluorescência/métodos , Testículo
10.
Vet Parasitol ; 309: 109773, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35930933

RESUMO

Essentially all grazing horses are infected with cyathostomin parasites. Adult cyathostomins reside in the large intestine of the horse and larval stages encyst within intestinal mucosa. Manual worm collection from aliquots of intestinal content is the current gold standard for retrieval and enumeration of luminal parasites, however, no research has been conducted to standardize specific parameters for processing and storage of samples. The aims of this study were (1) to evaluate the precision of current standard operating procedures for enumeration of luminal adult cyathostomin populations, (2) investigate the influence of chosen fixative, either 70 % ethanol or 10 % buffered formalin, as well as storage duration, immediately post necropsy vs. stored for eight weeks, on the magnitude and precision of worm counts, and (3) compare the luminal count magnitude between the three intestinal segments (cecum, ventral colon, dorsal colon). Ten miniature horses were enrolled in this study for euthanasia and necropsy over a four-week period. Luminal worm counts were conducted for 2 % aliquots of the cecum, ventral colon, and dorsal colon and samples were allocated to the two fixatives and the two storage durations. Precision was evaluated by coefficient of variation (CV) and was 13.04 % for total cyathostomin counts. Mean CV for large intestinal segments ranged from 15.31 % to 52.50 % irrespective of fixative used or storage duration. cecum worm counts were significantly lower compared to the ventral colon (p = 0.008) and dorsal colon (p = 0.01). Fixative and storage duration were not statistically associated with count precision or magnitude. This study demonstrated moderate to high precision estimates for luminal cyathostomin worm counts but did not identify any effects of fixative and storage duration within the framework of the study. This is the first study to determine cyathostomin worm count precision, and results will be useful for power analyses in the future.


Assuntos
Doenças dos Cavalos , Infecções Equinas por Strongyloidea , Animais , Ceco , Colo , Fezes/parasitologia , Fixadores , Doenças dos Cavalos/parasitologia , Cavalos , Intestino Grosso/parasitologia , Contagem de Ovos de Parasitas/veterinária , Infecções Equinas por Strongyloidea/parasitologia
11.
Ultramicroscopy ; 241: 113600, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35988477

RESUMO

Muscle samples are commonly chemically fixed or frozen immediately upon collection for biochemical and morphological analysis. Certain fixatives such as glutaraldehyde and osmium tetroxide are widely used for transmission electron microscopy (TEM) and lead to adequate preservation of muscle ultrastructure, but do not preserve the molecular features of samples. Methacarn is suggested to be a preferable chemical fixative for light microscopy because it maintains immunohistological features of samples. However, the efficacy of methacarn to preserve ultrastructural features as a primary chemical fixative for TEM is currently unclear. Additionally, cryo-preservation of samples for TEM analysis involves freezing processes such as plunge freezing, slam freezing, or high pressure freezing. High pressure freezing is the considered the gold standard but requires costly equipment and may not be a viable option for many labs collecting tissue samples from remote locations. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant that may allow for better structural preservation of samples by impairing ice damage that occurs during plunge/snap freezing. We aimed to assess the effectiveness of methacarn as a primary chemical fixative and determine the effect of pre-coating samples with DMSO before plunge/snap freezing tissues to be prepared for TEM. The micrographs of the methcarn-fixed samples indicate a loss of Z-disk integrity, intermyofibrillar space, mitochondria structure, and lipids. Ultimately, methacarn is not a viable primary fixative for tissue sample preparation for TEM. Similarly, liquid nitrogen freezing of samples wrapped in aluminum foil produced non-uniform Z-disk alignments that appeared smeared with swollen mitochondria. DMSO coating before freezing appears to lessen the alterations to contractile and mitochondrial morphological structures. DMSO appears to be useful for preserving the ultrastructure of sarcomeres if samples are covered before freezing.


Assuntos
Dimetil Sulfóxido , Tetróxido de Ósmio , Ácido Acético , Alumínio , Clorofórmio , Criopreservação , Fixadores/farmacologia , Glutaral , Gelo , Metanol , Microscopia Eletrônica de Transmissão , Músculos
12.
Protist ; 173(5): 125905, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36027633

RESUMO

Loxodes is one of the best ecologically characterized ciliate genera with numerous intriguing physiological abilities, including gravity-sensing organelles and nitrate respiration. However, these cells have been considered challenging to cultivate in bulk, and are poorly preserved by conventional fixatives used for fluorescence microscopy. Here we describe methods to grow and harvest Loxodes cells in bulk with liquid soil extract medium, as well as a new fixative called ZFAE (zinc sulfate, formaldehyde, acetic acid, ethanol) that can fix Loxodes cells more effectively than buffered formaldehyde or methanol. We show that ZFAE is compatible with immunofluorescence and the nuclear stain DAPI. Loxodes is thus now amenable to long-term maintenance, large-scale growth, and modern cell biology investigations of monoclonal strains in laboratory conditions.


Assuntos
Cilióforos , Metanol , Fixadores , Nitratos , Sulfato de Zinco , Formaldeído , Microscopia de Fluorescência , Etanol , Solo
13.
Anat Histol Embryol ; 51(6): 740-745, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35964229

RESUMO

Although formaldehyde is the most widely used and largely available fixative for preserving cadavers through decomposition prevention, it promotes darkening and weight gain, in addition to being considered carcinogenic. Ethyl alcohol has been proven a potential substitute to formaldehyde due to its effectiveness in tissue penetration, thus preventing proliferation of microorganisms; however, it can only be used alone for fixation of small parts. In view of such fixatives limitations, saturated salt solution has been widely employed based on its antimicrobial effect and ability to maintain tissue similar to the original one, in addition to exerting no hazardous effects as there is no evaporation of harmful substances. This research aimed to observe anatomical brain behaviour submitted to formaldehyde, alcohol, and saturated salt solution as fixatives. Fixatives were tested in 15 adult Wistar rats' brain, submerged in 10 ml of intended solution after removal for 4 weeks. Weight of the brains fixed in saturated salt did not change over the weeks. However, the weight of formaldehyde-fixed brains increased and the weight in alcohol-fixed brains decreased; in addition, modifications in all solutions measures were also observed. Alcohol provides a peculiar dehydrating effect as formaldehyde clearly increases the length of the pieces. Thus, since the saturated salt solution showed no important adjustment over the experimental time, it proved an efficient alternative for replacing formaldehyde and alcohol as fixative solutions of anatomical study of the brain.


Assuntos
Etanol , Formaldeído , Ratos , Animais , Fixadores/farmacologia , Ratos Wistar , Formaldeído/farmacologia , Etanol/farmacologia , Encéfalo , Fixação de Tecidos/veterinária
14.
N Biotechnol ; 71: 30-36, 2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-35878783

RESUMO

Most tissues in clinical practice are formalin-fixed and paraffin-embedded for histological as well as molecular analyses. The reproducibility and uniformity of molecular analyses is strictly dependent on the quality of the biomolecules, which is highly influenced by pre-analytical processes. In this study, the effect of different fixatives was compared, including formalin, Bouin's solution, RCL2® and TAG-1™ fixatives, by stringent application of ISO standards in mouse liver tissue processing, including formalin-free transport of tissues and tissue grossing in a refrigerated environment. The effect of fixatives was studied in terms of nucleic acid quality at the time of tissue processing and after one year of tissue storage at room temperature in the dark. Furthermore, a microcomputed tomography (CT) scan analysis was applied to investigate the paraffin embedding. The results show that the application of ISO standards in tissue processing allows analysis of 400 bases amplicons from RNA and 1000 bases from DNA, even in extracts from formalin-fixed and paraffin-embedded tissues. However, after one year storage at room temperature in the dark, a degradation of the nucleic acids was observed. Nevertheless, extracts can still be analyzed, but for metachronous tests it is highly recommended to repeat the quantitation of housekeeping genes in order to standardize the extent of nucleic acid degradation.


Assuntos
Formaldeído , Ácidos Nucleicos , Animais , Fixadores , Camundongos , Ácidos Nucleicos/genética , Padrões de Referência , Reprodutibilidade dos Testes , Fixação de Tecidos/métodos , Microtomografia por Raio-X
15.
Clin Anat ; 35(7): 987-997, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35879645

RESUMO

In 1992, Walter Thiel described and embalming method that rendered "lifelike" tissues. Over the last 30 years, the Thiel method has been introduced worldwide for medical training and scientific purposes. This review examines research which can be linked to the use of Thiel embalming. A systematic review was performed to identify articles published in the following categories: research content, disciplines involved, sources and quantities of tissues deployed, and changes in research scope related to changes in the chemical composition of Thiel embalming. Four-hundred twenty-four publications were included. A number of adaptations to the original Thiel protocol were found, aiming to provide suitable tissue-substitutes in the development of emerging medical technologies or procedures. Musculoskeletal surgery, anesthesia and intensive care were the most common disciplines that used Thiel embalmed tissues for research. Anatomy and biomechanics played a lesser role. An increase over time was observed in research outputs related to the Thiel method, while the number of specimens used per study decreased. The main centers using Thiel embalming were in Graz, Dundee, Sapporo, Bern, Zurich and Ghent, which jointly accounted for more than 54% of all research conducted using this method. Following three decades of use, the Thiel method has evolved into being a well-established embalming technique for research purposes. Its future is challenged by the demanding requirements on both technical facilities and personnel, limitations of certain chemicals for use as fixatives, costs, and questions as to how "lifelike" the embalmed-tissues are from an objective standpoint, all of which warrants future investigations.


Assuntos
Pesquisa Biomédica , Embalsamamento , Fenômenos Biomecânicos , Cadáver , Embalsamamento/métodos , Fixadores , Humanos
16.
STAR Protoc ; 3(3): 101524, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-35810413

RESUMO

This protocol describes how to characterize α-Smooth muscle actin (αSMA) spatiotemporal expression during mouse small intestinal development. Specific tissue fixation preserves αSMA arrangement in low αSMA expressing cells that are conventionally undetectable under αSMA immunofluorescent stain due to inappropriate fixative-caused artificial actin depolymerization. Parallel analysis of αSMA carbonylation allows estimation of oxidative damage in gut muscular lineage. This approach improves the molecular specificity offered by commercialized kits that estimate total protein carbonyl level in cell lysates without protein specificity. For complete details on the use and execution of this protocol, please refer to Hu et al. (2021).


Assuntos
Actinas , Músculo Liso , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Fixadores/metabolismo , Intestinos , Camundongos , Músculo Liso/metabolismo , Estresse Oxidativo
17.
Sci Rep ; 12(1): 12395, 2022 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-35858968

RESUMO

Fluorescence in situ hybridisation (FISH) is a powerful molecular technique that enables direct visualisation of specific bacterial species. Few studies have established FISH protocols for tonsil tissue in Carnoy's fixative, accordingly limiting its application to investigate the pathogenesis of tonsillar hyperplasia. Tonsil tissue from 24 children undergoing tonsillectomy for either recurrent tonsillitis or sleep-disordered breathing were obtained during a previous study. The specificity of each of the five FISH probes (Fusobacterium spp., Bacteroides spp., Streptococcus spp., Haemophilus influenzae and Pseudomonas spp.) were successfully optimised using pure and mixed bacterial isolates, and in Carnoy's fixed tonsil tissue. Bacteroides spp. were present in 100% of patients with microcolonies. In comparison, the prevalence of Fusobacterium spp. was 93.8%, Streptococcus spp. 85.7%, H. influenzae 82.35% and Pseudomonas spp. 76.5%. Notable differences in the organisation of bacterial taxa within a single microcolony were also observed. This is the first study to establish a robust FISH protocol identifying multiple aerobic and anaerobic bacteria in Carnoy's fixed tonsil tissue. This protocol provides a strong foundation for combining histological and microbiological analyses of Carnoy's fixed tonsil samples. It may also have important implications on the analysis of microorganisms in other human tissues prepared using the same techniques.


Assuntos
Tonsilectomia , Tonsilite , Bactérias/genética , Criança , Fixadores , Haemophilus influenzae , Humanos , Hibridização in Situ Fluorescente , Tonsila Palatina/patologia , Streptococcus , Tonsilite/patologia
18.
Virchows Arch ; 481(3): 335-350, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35857102

RESUMO

Biomarker testing is crucial for treatment selection in advanced non-small cell lung cancer (NSCLC). However, the quantity of available tissue often presents a key constraint for patients with advanced disease, where minimally invasive tissue biopsy typically returns small samples. In Part 1 of this two-part series, we summarise evidence-based recommendations relating to small sample processing for patients with NSCLC. Generally, tissue biopsy techniques that deliver the greatest quantity and quality of tissue with the least risk to the patient should be selected. Rapid on-site evaluation can help to ensure sufficient sample quality and quantity. Sample processing should be managed according to biomarker testing requirements, because tissue fixation methodology influences downstream nucleic acid, protein and morphological analyses. Accordingly, 10% neutral buffered formalin is recommended as an appropriate fixative, and the duration of fixation is recommended not to exceed 24-48 h. Tissue sparing techniques, including the 'one biopsy per block' approach and small sample cutting protocols, can help preserve tissue. Cytological material (formalin-fixed paraffin-embedded [FFPE] cytology blocks and non-FFPE samples such as smears and touch preparations) can be an excellent source of nucleic acid, providing either primary or supplementary patient material to complete morphological and molecular diagnoses. Considerations on biomarker testing, reporting and quality assessment are discussed in Part 2.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Ácidos Nucleicos , Biomarcadores , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/patologia , Prova Pericial , Fixadores , Formaldeído , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Inclusão em Parafina , Fixação de Tecidos/métodos
19.
Int. j. morphol ; 40(3): 566-572, jun. 2022. ilus, tab
Artigo em Espanhol | LILACS-Express | LILACS | ID: biblio-1385673

RESUMO

RESUMEN: La solución de formol es utilizada en las Escuelas de medicina como medio de fijación y conservación de cadáveres para el estudio de la Anatomía, a la que están expuestos estudiantes, técnicos y personal docente; es alergénica e irritante a las mucosas, y reconocida carcinogénica en humanos por International Agency for Research on Cancer (2006). El objetivo del presente estudio fue comparar resultados cuantitativos y cualitativos entre corazones de Gallus gallus domesticus, luego de aplicarles soluciones con y sin formol. Se formaron dos grupos al azar, a uno se le aplicó solución de formol al 10 %, y al otro solución libre de formol. Se realizaron medidas antropométricas, organolépticas, y de fotografía (Pretest, durante y Postest). Se elaboró base datos en Microsoft Excel (2019), y su procesamiento en SPSS Statistics 2017 Versión 25. Para variables cuantitativas se aplicó la prueba de Shapiro-Wilk, y t-Student pareada. Para variables cualitativas el test Alfa de Cronbach, Chi cuadrado (X2) y los correspondientes coeficientes de asociación (D de Somers y Tau b de Kendal). Los resultados obtenidos de las variables peso, largo, y altura presentaron diferencia estadística significativa (p-valor <0,05), siendo diferente para el ancho y grosor de la pared del ventrículo izquierdo. Las variables color y consistencia presentaron diferencias significativa (p-valor <0,05). El olor irritante a las mucosas estuvo presente durante todo el estudio con la solución con formol. A la inspección, ninguno de los dos grupos presento colonización - descomposición. Se concluye que, los órganos en experimentación que se les aplicó solución libre de formol, presentaron mejores resultados con respecto a los que se les aplico formol al 10 %.


SUMMARY: The formaldehyde solution is used in medical schools as a means of fixing and preserving corpses for the study of Anatomy, to which students, technicians and teaching personnel are exposed; it is allergenic and irritant to the mucosa, and recognized as a human carcinogen by the International Agency for Research on Cancer (2006). The objective of the present study was to compare quantitative and qualitative results between Gallus gallus domesticus hearts, after applying solutions with and without formaldehyde. Two groups were formed at random, to one a 10 % formaldehyde solution was applied, and to the other formaldehyde- free solution. Anthropometric, organoleptic, and photographic measurements were carried out (Pretest, during and Posttest). A database was prepared in Microsoft Excel (2019), and its processing in SPSS Statistics 2017 Version 25. For quantitative variables, the Shapiro-Wilk test and t-Student paired were applied. For qualitative variables the Cronbach's Alpha test, Chi square (X2) and the corresponding association coefficients (Somers D and Kendal's Tau b). The results obtained from the variables weight, length, and height presented a statistically significant difference (p-value <0.05), being different for the width and thickness of the left ventricular wall. The variables color and consistency showed significant differences (p-value <0.05). The irritating smell to the mucous membranes was present throughout the study with the formaldehyde solution. Upon inspection, neither group showed colonization - decomposition. It is concluded that the organs in experimentation that were applied formaldehyde-free solution presented better results compared to those that were applied 10 % formaldehyde.


Assuntos
Animais , Soluções/administração & dosagem , Preservação de Tecido/métodos , Fixadores/farmacologia , Formaldeído/administração & dosagem , Coração/efeitos dos fármacos , Preservação de Órgãos , Galinhas , Antropometria
20.
Commun Biol ; 5(1): 487, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35595960

RESUMO

Chemical fixations have been thought to preserve the structures of the cells or tissues. However, given that the fixatives create crosslinks or aggregate proteins, there is a possibility that these fixatives create nanoscale artefacts by aggregation of membrane proteins which move around freely to some extent on the cell surface. Despite this, little research has been conducted about this problem, probably because there has been no method for observing cell surface structures at the nanoscale. In this study, we have developed a method to observe cell surfaces stably and with high resolution using atomic force microscopy and a microporous silicon nitride membrane. We demonstrate that the size of the protrusions on the cell surface is increased after treatment with three commonly used fixatives and show that these protrusions were created by the aggregation of membrane proteins by fixatives. These results call attention when observing fixed cell surfaces at the nanoscale.


Assuntos
Artefatos , Proteínas de Membrana , Membrana Celular , Fixadores , Microscopia de Força Atômica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...