Cell Surface Immunofluorescence Staining Protocol v1

protocols.io ◽  
2016 ◽  
Author(s):  
BioLegend not provided ◽  
Inc not provided
Keyword(s):  
Cell Surface ◽  
Immunofluorescence Staining ◽  
Staining Protocol

scholarly journals MiR-26a-5p inhibits GSK3β expression and promotes cardiac hypertrophy in vitro

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10371
Author(s):  
Liqun Tang ◽  
Jianhong Xie ◽  
Xiaoqin Yu ◽  
Yangyang Zheng
Keyword(s):  
Cell Surface ◽  
Cardiac Hypertrophy ◽  
Surface Area ◽  
Myocardial Cell ◽  
Immunofluorescence Staining ◽  
Cell Surface Area ◽  
Direct Target

Background The role of miR-26a-5p expression in cardiac hypertrophy remains unclear. Herein, the effect of miR-26a-5p on cardiac hypertrophy was investigated using phenylephrine (PE)-induced cardiac hypertrophy in vitro and in a rat model of hypertension-induced hypertrophy in vivo. Methods The PE-induced cardiac hypertrophy models in vitro and vivo were established. To investigate the effect of miR-26a-5p activation on autophagy, the protein expression of autophagosome marker (LC3) and p62 was detected by western blot analysis. To explore the effect of miR-26a-5p activation on cardiac hypertrophy, the relative mRNA expression of cardiac hypertrophy related mark GSK3β was detected by qRT-PCR in vitro and vivo. In addition, immunofluorescence staining was used to detect cardiac hypertrophy related mark α-actinin. The cell surface area was measured by immunofluorescence staining. The direct target relationship between miR-26a-5p and GSK3β was confirmed by dual luciferase report. Results MiR-26a-5p was highly expressed in PE-induced cardiac hypertrophy. MiR-26a-5p promoted LC3II and decreased p62 expression in PE-induced cardiac hypertrophy in the presence or absence of lysosomal inhibitor. Furthermore, miR-26a-5p significantly inhibited GSK3β expression in vitro and in vivo. Dual luciferase report results confirmed that miR-26a-5p could directly target GSK3β. GSK3β overexpression significantly reversed the expression of cardiac hypertrophy-related markers including ANP, ACTA1 and MYH7. Immunofluorescence staining results demonstrated that miR-26a-5p promoted cardiac hypertrophy related protein α-actinin expression, and increased cell surface area in vitro and in vivo. Conclusion Our study revealed that miR-26a-5p promotes myocardial cell autophagy activation and cardiac hypertrophy by regulating GSK3β, which needs further research.

scholarly journals cFos immunoreactivity is enhanced with biotin amplification.

1994 ◽  
Vol 42 (12) ◽  
pp. 1635-1642 ◽  
Author(s):  
K A Berghorn ◽  
J H Bonnett ◽  
G E Hoffman
Keyword(s):  
Signal To Noise Ratio ◽  
Immediate Early Gene ◽  
Immunofluorescence Staining ◽  
Early Gene ◽  
Primary Antibody ◽  
Immediate Early ◽  
High Signal ◽  
Fluorescence Staining ◽  
Conventional Procedure ◽  
Staining Protocol

Through modification of the protocol by Adams, we developed a biotin amplification procedure for immunofluorescence staining of immediate early gene proteins and also applied biotin amplification for metal enhancement of diaminobenzidine staining in an immunoperoxidase protocol. Commercially available anti-cFos antisera were used to compare conventional "Elite" avidin-biotin complex reactions with biotin amplification reactions (visualized with peroxidase staining or streptavidin-Texas Red fluorescence). Biotin amplification and peroxidase staining (with or without nickel salts) enabled detection of cFos in stimulated neurons with primary antibody concentrations five- to tenfold lower than the conventional procedure. With immunofluorescence staining, at primary antibody concentrations too low to detect cFos with the conventional biotin-streptavidin fluorescence staining protocol, biotin amplification enabled clear cFos fluorescence staining with both antisera. The fluorescence staining exhibited a high signal-to-noise ratio and enabled antibody concentrations four times lower than those used for conventional ABC "Elite" peroxidase procedures. In conclusion, the application of biotin amplification to cFos immunocytochemical localization has the promise of aiding the scientist in detecting these immediate early gene products.

scholarly journals Optimized immunofluorescence staining protocol for imaging germinal centers in secondary lymphoid tissues of vaccinated mice

2021 ◽  
Vol 2 (3) ◽  
pp. 100499
Author(s):  
Sigrid Fra-Bido ◽  
Simon A. Walker ◽  
Silvia Innocentin ◽  
Michelle A. Linterman
Keyword(s):  
Germinal Centers ◽  
Immunofluorescence Staining ◽  
Lymphoid Tissues ◽  
Staining Protocol

γ-COPI mediates the retention of kAE1 G701D protein in Golgi apparatus – a mechanistic explanation of distal renal tubular acidosis associated with the G701D mutation

2017 ◽  
Vol 474 (15) ◽  
pp. 2573-2584 ◽  
Author(s):  
Natapol Duangtum ◽  
Mutita Junking ◽  
Suratchanee Phadngam ◽  
Nunghathai Sawasdee ◽  
Andrea Castiglioni ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
Renal Tubular Acidosis ◽  
Mechanistic Explanation ◽  
Distal Renal Tubular Acidosis ◽  
Immunofluorescence Staining ◽  
Cell Surface Expression ◽  
Small Interference Rna ◽  
Golgi Retention ◽  
Renal Tubular

Mutations of the solute carrier family 4 member 1 (SLC4A1) gene encoding kidney anion (chloride/bicarbonate ion) exchanger 1 (kAE1) can cause genetic distal renal tubular acidosis (dRTA). Different SLC4A1 mutations give rise to mutant kAE1 proteins with distinct defects in protein trafficking. The mutant kAE1 protein may be retained in endoplasmic reticulum (ER) or Golgi apparatus, or mis-targeted to the apical membrane, failing to display its function at the baso-lateral membrane. The ER-retained mutant kAE1 interacts with calnexin chaperone protein; disruption of this interaction permits the mutant kAE1 to reach the cell surface and display anion exchange activity. However, the mechanism of Golgi retention of mutant kAE1 G701D protein, which is otherwise functional, is still unclear. In the present study, we show that Golgi retention of kAE1 G701D is due to a stable interaction with the Golgi-resident protein, coat protein complex I (COPI), that plays a role in retrograde vesicular trafficking and Golgi-based quality control. The interaction and co-localization of kAE1 G701D with the γ-COPI subunit were demonstrated in human embryonic kidney (HEK-293T) cells by co-immunoprecipitation and immunofluorescence staining. Small interference RNA (siRNA) silencing of COPI expression in the transfected HEK-293T cells increased the cell surface expression of transgenic kAE1 G701D, as shown by immunofluorescence staining. Our data unveil the molecular mechanism of Golgi retention of kAE1 G701D and suggest that disruption of the COPI-kAE1 G701D interaction could be a therapeutic strategy to treat dRTA caused by this mutant.

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