Glyoxal Fixation Is Optimal for Immunostaining of Brain Vessels, Pericytes and Blood-Brain Barrier Proteins

Thomas, Sithara; Sadanandan, Jayanarayanan; Blackburn, Spiros; McBride, Devin W.; Dienel, Ari; Hong, Sung‐Ha; Zeineddine, Hussein A.; Pandit, Peeyush Kumar Thankamani

doi:10.3390/ijms23147776

Cited by 6 publications

(4 citation statements)

References 47 publications

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“…To simultaneously observe DNA and other biomolecules, such as mRNAs, cell fixation is required. ,, Fixation is also required to obtain the locations of molecules with fast dynamics, such as transcription factors, with a high resolution owing to the trade-off between spatial resolution and temporal resolution. , Paraformaldehyde (PFA) has been generally used as a cross-linking fixation method, and fixation using glyoxal has recently been introduced . Each method has its own advantages and disadvantages depending on the types of cells or proteins. − In this study, we tested both fixation methods to determine whether the gene loci remained visible in fixed E. coli cells.…”

Section: Resultsmentioning

confidence: 99%

Analysis of Fluorescent Proteins for Observing Single Gene Locus in a Live and Fixed Escherichia coli Cell

Son,

Kim,

Yang

et al. 2024

J. Phys. Chem. B

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Fluorescent proteins (FPs) are essential tools for advanced microscopy techniques such as super-resolution imaging, single-particle tracking, and quantitative single-molecule counting. Various FPs fused to DNA-binding proteins have been used to observe the subcellular location and movement of specific gene loci in living and fixed bacterial cells. However, quantitative assessments of the properties of FPs for gene locus measurements are still lacking. Here, we assessed various FPs to observe specific gene loci in live and fixed Escherichia coli cells using a fluorescent repressor-operator binding system (FROS), tet operator-Tet repressor proteins (TetR). Tsr-fused FPs were used to assess the intensity and photostability of various FPs (five red FPs: mCherry2, FusionRed, mRFP, mCrimson3, and dKatushka; and seven yellow FPs: SYFP2, Venus, mCitrine, YPet, mClover3, mTopaz, and EYFP) at the single-molecule level in living cells. These FPs were then used for gene locus measurements using FROS.Our results indicate that TetR-mCrimson3 (red) and TetR-EYFP (yellow) had better properties for visualizing gene loci than the other TetR-FPs. Furthermore, fixation procedures affected the clustering of diffusing TetR-FPs and altered the locations of the TetR-FP foci. Fixation with formaldehyde consistently disrupted proper DNA locus observations using TetR-FPs. Notably, the foci measured using TetR-mCrimson3 remained close to their original positions in live cells after glyoxal fixation. This in vivo study provides a cell-imaging guide for the use of FPs for gene-locus observation in E. coli and a scheme for evaluating the use of FPs for other cell-imaging purposes.

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Section: Resultsmentioning

confidence: 99%

Analysis of Fluorescent Proteins for Observing Single Gene Locus in a Live and Fixed Escherichia coli Cell

Son,

Kim,

Yang

et al. 2024

J. Phys. Chem. B

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“…Immunostaining: One sham and one SAH male C57BL/6 mouse were euthanized on day 5 via cardiac perfusion of PBS and the brains were stored in 3% glyoxal until processing. 25 Brains were sectioned into 40µm thick slices using a vibratome (Leica VT 1000S). Brain slices (at -2 from bregma) were stained for 12-LOX (1:100, sc-365194, Santa Cruz), 15-LOX (1:100, sc-133085, Santa Cruz), and laminin (1:200, sc-59854, Santa Cruz).…”

Section: Elisamentioning

confidence: 99%

12/15-Lipooxygenase Inhibition Reduces Microvessel Constriction and Microthrombi after Subarachnoid Hemorrhage in Mice

Dienel,

Hong,

Zeineddine

et al. 2024

Preprint

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Background and Purpose Impaired cerebral circulation, induced by blood vessel constrictions and microthrombi, leads to delayed cerebral ischemia after subarachnoid hemorrhage (SAH). 12/15-Lipooxygenase (12/15-LOX) overexpression has been implicated in worsening early brain injury outcomes following SAH. However, it is unknown if 12/15-LOX is important in delayed pathophysiological events after SAH. Since 12/15-LOX produces metabolites that induce inflammation and vasoconstriction, we hypothesized that 12/15-LOX leads to microvessel constriction and microthrombi formation after SAH, and thus 12/15-LOX is an important target to prevent delayed cerebral ischemia. Methods SAH was induced in C57BL/6 and 12/15-LOX−/− mice of both sexes by endovascular perforation. Expression of 12/15-LOX was assessed in brain tissue slices and in vitro. C57BL/6 mice were administered either ML351 (12/15-LOX inhibitor) or vehicle. Mice were evaluated for daily neuroscore and euthanized on day five to assess cerebral 12/15-LOX expression, vessel constrictions, platelet activation, microthrombi, neurodegeneration, infarction, cortical perfusion, and for development of delayed deficits. Finally, the effect of 12/15-LOX inhibition on platelet activation was assessed in SAH patient samples using a platelet spreading assay. Results In SAH mice, 12/15-LOX was upregulated in brain vascular cells and there was an increase in 12-S-HETE. Inhibition of 12/15-LOX improved brain perfusion on days 4–5 and attenuated delayed pathophysiological events, including microvessel constrictions, microthrombi, neuronal degeneration, and infarction. Additionally, 12/15-LOX inhibition reduced platelet activation in human and mouse blood samples. Conclusions Cerebrovascular 12/15-LOX overexpression plays a major role in brain dysfunction after SAH by triggering microvessel constrictions and microthrombi formation, which reduces brain perfusion. Inhibiting 12/15-LOX may be a therapeutic target to improve outcomes after SAH.

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“…Alcohol fixation is another type of fixators successfully used for intracellular staining in flow cytometry [10], but it results in cell clumping and loss of light scatter thus it is completely unacceptable for cell fixation and storage [11]. Less popular among fixators is glyoxal-based solutions, which is nowadays positioned as alternative fixator to paraformaldehyde in immunostaining [12,13]. Authors emphasize glyoxal's advantages including safeness and easiness in handling, better preservation of cell fine structure and more intense immunostaining without antigen retrieval.…”

Section: Introductionmentioning

confidence: 99%

How fixation affects the results of lymph node immunophenotyping by flow cytometry

Yerpasheva,

Kemaykin,

Zhunis

et al. 2023

J CLIN MED KAZ

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Aim: Flow cytometric diagnosis of lymphoma and leukemia is of high clinical and research importance. However, performing flow cytometry analysis on the day of biopsy might be of challenge due to several reasons, including late sample delivery, problems of preparing the reliable panel for immunophenotyping based on other diagnostic studies, etc. This problem could be partially solved if cell suspension could be fixed and stained on another day or after several days after standard FFPE (formalin-fixed and paraffin-embedded) procedure. Material and methods: Addressing this issue, we compared staining of live lymphocytes in suspension obtained from lymph node biopsies and same specimens fixed using 2-4%-paraformaldehyde, 1-3%-glyoxal, and 0.1-1% glutaraldehyde with subsequent immunostaining on the next day or later. Results: Staining after fixation could be partially representative only after paraformaldehyde fixation for 20 min and subsequent storage of cell suspension in phosphate-buffer saline within not more than 3 days. Probes stained after fixation always shows lower stain index compared to staining of live cells. Conclusion: Staining after fixation cannot be used for determining of the percentage of CD45-positive cells and for testing B-cell lymphomas since antigens against light chains of IgG cannot be properly detected in fixed specimens.

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Glyoxal Fixation Is Optimal for Immunostaining of Brain Vessels, Pericytes and Blood-Brain Barrier Proteins

Cited by 6 publications

References 47 publications

Analysis of Fluorescent Proteins for Observing Single Gene Locus in a Live and Fixed Escherichia coli Cell

Analysis of Fluorescent Proteins for Observing Single Gene Locus in a Live and Fixed Escherichia coli Cell

12/15-Lipooxygenase Inhibition Reduces Microvessel Constriction and Microthrombi after Subarachnoid Hemorrhage in Mice

How fixation affects the results of lymph node immunophenotyping by flow cytometry

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