scholarly journals Protein-retention expansion microscopy for visualizing subcellular organelles in fixed brain tissue

Author(s):  
Logan A. Campbell ◽  
Katy E. Pannoni ◽  
Niesha A. Savory ◽  
Dinesh Lal ◽  
Shannon Farris
2020 ◽  
Author(s):  
Logan A. Campbell ◽  
Katy E. Pannoni ◽  
Niesha A. Savory ◽  
Dinesh Lal ◽  
Shannon Farris

ABSTRACTProtein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduces fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals. Here we describe a proExM workflow optimized for resolving subcellular organelles (mitochondria and the Golgi apparatus) and reporter-labeled spines in fixed mouse brain tissue. By directly comparing proExM staining and digestion protocols, we found that immunostaining before proExM and using a Proteinase K based digestion for 8 hours consistently resulted in the best fluorescence signal to resolve subcellular organelles while maintaining sufficient reporter labeling to visualize spines and trace individual neurons. With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in reconstructed CA2 neurons of the hippocampus.


2006 ◽  
Vol 37 (03) ◽  
Author(s):  
R Husain ◽  
HS Fink ◽  
K Lang ◽  
B Merkle ◽  
R Bauer ◽  
...  
Keyword(s):  

2014 ◽  
Vol 37 (2) ◽  
pp. 85 ◽  
Author(s):  
Ata Topcuoglu ◽  
Mustafa Albayrak ◽  
Hayriye Erman ◽  
Huriye Balci ◽  
Mesut Karakus ◽  
...  

Purpose: The purpose of this study was to analyze the effects of estrogen deficiency and hormone replacement therapy (HRT) on fibrinolytic activity in a rat mode of surgically-induced menopause. Methods: Twelve-week-old, sexually mature female Sprague-Dawley rats, each weighing 200–250 g, were randomly divided into four groups: (1) sham-operated group, (2) ovariectomy group, (3) ovariectomy group followed by oral administration of daily 17β-estradiol (0.02 mg/kg/day) (E2) + norethisterone acetate (0.01 mg/kg/day), and (4) ovariectomy group followed by oral administration of daily 17β-estradiol (0.01 mg/kg/day) + drospirenone (0.02 mg/kg/day). Tissue plasminogen activator (tPA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1/tPA levels were measured as markers of fibrinolysis in plasma and liver and brain tissue. Results: Compared with sham-operated rats, ovariectomized rats showed higher levels of fibrinolytic activity; however, the increased fibrinolytic activity in plasma and liver tissue was significantly reduced by HRT regimens. No change was observed in the levels of fibrinolytic activity in brain tissue. Conclusions: HRT showed beneficial effects by decreasing fibrinolytic activity related to surgically-induced menopause. Short-term HRT treatment was associated with a shift in the procoagulant-anticoagulant balance toward a procoagulant state.


2019 ◽  
Author(s):  
Huaimin Wang ◽  
Zhaoqianqi Feng ◽  
Weiyi Tan ◽  
Bing Xu

<p>Selectively targeting cell nucleolus remains a challenge. Here we report the first case that D-peptides form membraneless molecular condensates with RNA for targeting cell nucleolus. A D-peptide derivative, enriched with lysine and hydrophobic residues, self-assembles to form nanoparticles, which enter cells through clathrin dependent endocytosis and mainly accumulate at cell nucleolus. Structural analogue of the D-peptide reveals that particle morphology of the assemblies, which depends on the side chain modification, favors the cellular uptake. Contrasting to those of the D-peptide, the assemblies of the corresponding L-enantiomer largely localize in cell lysosomes. Preliminary mechanism study suggests that the D-peptide nanoparticles interact with RNA to form membraneless condensates in the nucleolus, which further induces DNA damage and results in cell death. This work illustrates a new strategy for rationally designing supramolecular assemblies of D-peptides for targeting subcellular organelles.</p>


Author(s):  
A. G. Zhukova ◽  
L. G. Gorokhova ◽  
A. S. Kazitskaya ◽  
T. K. Yadykina ◽  
N. N. Mikhailova ◽  
...  

Introduction. Fluorine compounds in small doses, but with prolonged exposure, cause various disorders in organs at the cellular and molecular levels. Activation of free-radical processes plays an important role in the damaging eff ect of fl uorides. Th erefore, one of the most eff ective ways to limit fl uorine-induced damage is to directly aff ect free-radical processes using herbal preparations with antioxidant properties.The aim of the study is to study the eff ect of a dihydroquercetin-based drug on the activity of free radical processes in brain tissue under subchronic exposure to sodium fl uoride (NaF).Materials and methods. Th e work was performed on white male laboratory rats weighing 200-250 g. Th e rats were divided into 3 groups: 1 — control; 2 — rats with chronic exposure to sodium fl uoride (NaF) for 9 weeks; 3 — rats receiving a NAF solution with simultaneous administration of a complex drug based on dihydroquercetin at a dose of 3 mg/kg in 1% starch gel for 3, 6 and 9 weeks. The activity of free radical oxidation and antioxidant defense enzymes — superoxide dismutase (SOD) and catalase-was determined in the cerebral cortex. Th e level of expression of hypoxia-induced transcription factor HIF — 1A and inducible forms of proteins HSP72 and HSP32 were determined in the cytosolic fraction of brain tissue.Results. In the early stages of subchronic fl uoride exposure (1-3 weeks), the expression of protective proteins HIF-1α, HSP72, HSP32 and catalase was shown in the rat cortex, as a result of which the activity of free-radical processes was maintained at the control level. An increase in the timing of fl uoride intake to 9 weeks led to a decrease in antioxidant protection and signifi cant activation of free radical oxidation in brain tissue. Daily administration of a complex drug with dihydroquercetin for 3, 6 and 9 weeks to rats with subchronic fl uoride exposure led to a decrease in the severity of pro- and antioxidant balance disorders in the cerebral cortex. At the same time, the greatest protective eff ect of dihydroquercetin with fl uoride exposure was manifested by the 9th week of its administration.Conclusions. When subchronic intake of fl uorides in the body, the drug based on dihydroquercetin has a neuroprotective eff ect, which is manifested by an increase in the activity of antioxidant enzymes of fr ee radical oxidation and catalase and the resistance of the cortex to induced fr ee radical oxidation.


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