Advantages and Disadvantages of Zinc-Ethanol-Formaldehyde as a Fixative for Immunocytochemical Studies and Confocal Laser Microscopy

Коржевский, Д. Э.; Sukhorukova, E. G.; Gilerovich, E. G.; Петрова, Е. С.; Кирик, О. В.; Grigor’ev, I. P.

doi:10.1007/s11055-014-9948-8

Cited by 17 publications

(12 citation statements)

References 3 publications

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“…В настоящее время производится широкий спектр моноклональных антител к ТГ, а также поликлональных антител, которые позволяют выявлять этот фермент иммуногистохимически в нервной системе и других отделах организма человека и различных видов животных. В наших предыдущих исследованиях было показано, что [24,25].…”

Section: Immunohistochemical Markers For Neurobiologyunclassified

“…Показано, что высокое качество окраски парафиновых срезов тканей можно достичь при использовании протокола окраски, предложенного нами ранее [4]. Допустимы различные виды фиксации гистологического материала, однако оптимальные результаты окрашивания были получены при использовании цинк-этанол-формальдегида [24,25]. С помощью данного метода было изучено распределение холинергических нервных клеток спинного мозга (СМ) крысы на разных сроках эмбрионального развития, в раннем постнатальном периоде и у взрослых животных [3,40,41].…”

Section: Immunohistochemical Markers For Neurobiologyunclassified

“…В наших исследованиях применяли моноклональные мышиные антитела к белку SMI-32 (Bio Legend, США) для исследования нервных волокон седалищного нерва крысы в норме и после травмы. Реакцию проводили на парафиновых срезах после фиксации материала в цинк-этанол-формальдегиде [24,25].…”

Section: антитела Smi-32 против белков нейрофиламентовunclassified

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Immunohistochemical markers for neurobiology

Коржевский

Эдуардович²,

Grigor’ev

et al. 2020

Medical academic journal

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In neurobiological studies, crucial is the selection of most appropriate and informative experimental methods, one of which is immunohistochemistry. This review briefly summarizes the experience of adaptation of immunohistochemical methods to nervous system studies accumulated over years the Laboratory of Functional Morphology of the Central and Peripheral Nervous System (Institute of Experimental Medicine). The aim of this work was to determine the most effective and reliable immunomarkers for neurobiological studies. The article contains theoretical basis and practical recommendations for use of key cytospecific and functional markers used in studies of structural and functional organization of brain and spinal cord of mammalian animals and human. In particular, the results of immunohistochemical reactions to neural markers (NeuN, neurofilament proteins, alpha-tubulin, alpha-synuclein), neurotransmitter synthesizing enzymes (tyrosine hydroxylase, glutamate decarboxylase, choline acetyltransferase, NO synthase) and glial markers (GFAP, glutamine synthetase, Iba-1, vimentin) are demonstrated. The presented methodology is useful for experimental neurobiology and clinical morphological diagnostics.

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Section: Immunohistochemical Markers For Neurobiologyunclassified

Section: антитела Smi-32 против белков нейрофиламентовunclassified

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Immunohistochemical markers for neurobiology

Коржевский

Эдуардович²,

Grigor’ev

et al. 2020

Medical academic journal

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“…In the Laboratory of Morphology at the Institute of Experimental Medicine (Saint Petersburg), a new zinc-containing combination fixative -zinc-ethanolformaldehyde (1 g of zinc chloride in 96% ethanol and concentrated formalin, 9:1) -was developed [84]; that reagent demonstrated good preservation of the brain tissue and internal organs of humans and laboratory animals [85,86]. Immunohistochemical study of nervous tissue (definitive and embryonic) and peripheral organs fixed in zinc-ethanol-formaldehyde allowed for detecting a large number of proteins: calbindin, calretinin, choline acetyltransferase, glutamate decarboxylase, glial fibrillary acidic protein, Iba-1 and NeuN proteins, neuronspecific enolase, synaptophysin, tyrosine hydroxylase, alpha-tubulin, vimentin, nestin and others [87][88][89][90][91][92][93][94][95][96][97][98].…”

Section: Reviewsmentioning

confidence: 99%

Current Technologies for Fixation of Biological Material for Immunohistochemical Analysis (Review)

Grigor’ev¹,

Коржевский²

2018

Sovrem Tehnol Med

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Based on their own experience and published reports the authors provide an insight into the existing methods of fixation of biological material used in immunohistochemistry. The first quality of an immunohistochemical fixative should be its ability to preserve the tissue structure so that the antigenic properties of macromolecules are minimally affected. Considering this point, the review analyzes the applicability of commonly used fixatives to immunohistochemical staining; among those, aldehydes (formaldehyde, glutaraldehyde, glyoxal), dehydrating (coagulating) agents (ethanol, methanol, acetone), combined fixation solutions (Bouin's solution, Carnoy's solution, methacarn, etc.), as well as the recent zinc-containing fixatives and commercial products. Most of these fixatives inevitably change the tertiary and quaternary structure of many proteins; therefore, the detection of these proteins by immunohistochemistry requires an additional procedure of unmasking the epitopes using proteolytic enzymes or elevated temperatures. When compared for the preservation of antigenic structures, a high quality of the novel zinc-containing fixative-zinc-ethanol-formaldehyde-was noted. It has been concluded that none of the fixatives known to date has such a combination of properties that allow obtaining high-quality histological preparations and, at the same time, allows for detecting of any antigens in the stained tissue.

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“…Specimens were fi xed in zinc-ethanol-formaldehyde [2] and processed by standard methods. Sections of thickness 7-12 μm were cut from blocks.…”

mentioning

confidence: 99%

Detection of Glomeruli in the Human Cerebellum Using an Immunocytochemical Reaction for Synaptophysin and Confocal Laser Microscopy

Gilerovich

Sukhorukova

Кирик

et al. 2015

Neurosci Behav Physi

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The cerebellum controls coordination and motor activity via complex interactions between excitatory and inhibitory systems involving the intrinsic nuclei of the cerebellum [1]. The fi rst afferent component in this system consists of special synaptic formations -glomeruli -which are located in the granular layer of the cerebellar cortex. Glomeruli have a complex structure: their composition includes granule cell dendrites, mossy fi bers, and the axons of large stellate neurons (Golgi cells) [6], forming synaptic complexes. Studies of the structural organization of glomeruli are mainly performed using electron microscopy, which requires extensive specimen processing. Immunocytochemical detection of synaptophysin may be useful for the functional interpretation of the state of synaptic structures in cerebellar glomeruli, this being a glycoprotein and an essential part of the walls of synaptic vesicles. Synaptophysin is synthesized in the cytoplasm of neurons and is transported to synaptic terminals. Synaptophysin is therefore not infrequently used for studies of synaptogenesis [7]. An important property of synaptophysin is its high level of resistance to autolysis [8], which underlies its suitability for studying synaptic structures in humans, which has been confi rmed by data obtained from studies of the peripheral nervous system [3][4][5].The aim of the present work was to identify the potential for using immunocytochemical reaction for synaptophysin for analysis of the structural organization of cerebellar glomeruli in humans using light and confocal laser microscopy.Studies were performed using paraffi n blocks of human cerebellum (n = 8, age 20-62 years) from the collection of the Laboratory for the Functional Morphology of the Central and Peripheral Nervous System, Research Institute of Experimental Medicine. Specimens were fi xed in zinc-ethanol-formaldehyde [2] and processed by standard methods. Sections of thickness 7-12 μm were cut from blocks. Synaptophysin was detected using rabbit polyclonal antibodies to human synaptophysin (Spring Bioscience, USA; catalog No. E2174). Secondary reagents were from an EnVision+ HRP/DAB kit (Dako, Denmark) for light microscopy. For confocal laser microscopy, primary mouse monoclonal antibodies to synaptophysin, clone SY38 (Dako, Denmark; catalog No. M0776) were used, along with secondary biotinylated antibodies from an LSAB2 SystemThe aim of the present work was to develop methods for the detection of complex synaptic groups (glomeruli) in the human cerebellum using an immunocytochemical reaction for synaptophysin. The protocols presented here yield high-quality preparations for light and confocal laser microscopy on which individual glomeruli and intraglomerular axon terminals can be clearly identifi ed. These preparations are suitable for quantitative analysis.

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Advantages and Disadvantages of Zinc-Ethanol-Formaldehyde as a Fixative for Immunocytochemical Studies and Confocal Laser Microscopy

Cited by 17 publications

References 3 publications

Immunohistochemical markers for neurobiology

Immunohistochemical markers for neurobiology

Current Technologies for Fixation of Biological Material for Immunohistochemical Analysis (Review)

Detection of Glomeruli in the Human Cerebellum Using an Immunocytochemical Reaction for Synaptophysin and Confocal Laser Microscopy

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