tem fixation solution  (Servicebio Inc)

Journal: Cell Biology and Toxicology

Article Title: METTL3, m6A modification, and EGR1: interplay affecting myocardial I/R injury outcomes

doi: 10.1007/s10565-024-09937-7

Figure Lengend Snippet: The effect of EGR1 on cardiomyocyte mitophagy and cardiomyocyte injury in the H/R damage model. Note: ( A ) Schematic diagram of the extraction of primary cardiomyocytes and construction of the H/R damage model; ( B ) CCK-8 assay to measure the cell viability of cardiomyocytes in different treatment groups; ( C ) ELISA to measure the levels of cTn and CK-MB in the supernatant of cardiomyocytes in different treatment groups; ( D ) RT-qPCR to detect the mRNA expression of EGR1 in cardiomyocytes from different treatment groups; ( E ) Western blot to detect and quantify the protein expression of EGR1 in cardiomyocytes from different treatment groups; ( F ) TEM to observe the morphology of cardiomyocyte mitochondria in each group, with a scale bar of 500 nm and blue arrows indicating mitochondria; ( G ) Representative immunofluorescence images showing the co-localization of GFP-LC3B (green) and mitochondria (red) in cardiomyocytes from different treatment groups, with a scale bar of 25 μm, and quantification of the number of co-localized spots between GFP-LC3B and mitochondria, with DAPI (blue) indicating the nucleus; ( H ) Representative immunofluorescence images showing the co-localization of MTR-Green (green) and lysosomes (red) in cardiomyocytes from different treatment groups, with a scale bar of 50 μm, and quantification of the number of co-localized spots between lysosomes and mitochondria in each cell; ( I ) Western blot to detect and quantify the expression of mitophagy-related proteins in cardiomyocytes from different treatment groups; ( J ) Schematic diagram of shEGR1 transfection; ( K ) RT-qPCR to measure the expression of EGR1 in cardiomyocytes from different treatment groups; ( L ) Schematic diagram of 3-MA treatment after shEGR1 transfection in the H/R damage model; ( M ) TEM to observe the morphology of cardiomyocyte mitochondria in each group, with a scale bar of 500 nm and blue arrows indicating mitochondria; (N) Representative immunofluorescence images showing the co-localization of GFP-LC3B (green) and mitochondria (red) in cardiomyocytes from different treatment groups, with a scale bar of 25 μm, and quantification of the number of co-localized spots between GFP-LC3B and mitochondria, with DAPI (blue) indicating the nucleus; ( O ) Representative immunofluorescence images showing the co-localization of MTR-Green (green) and lysosomes (red) in cardiomyocytes from different treatment groups, with a scale bar of 50 μm, and quantification of the number of co-localized spots between lysosomes and mitochondria in each cell; ( P ) Western blot to detect and quantify the expression of mitophagy-related proteins in cardiomyocytes from different treatment groups; ( Q ) CCK-8 assay to measure the cell viability of cardiomyocytes in different treatment groups; ( R ) ELISA to measure the levels of cTnI and CK-MB in the supernatant of cardiomyocytes in different treatment groups. MTR: MitoTracker, LTR: LysoTracker; ** indicates a significant difference between two groups with p < 0.01, *** indicates a significant difference between two groups with p < 0.001, **** indicates a significant difference between two groups with p < 0.0001. All experiments were repeated three times

Article Snippet: The processed cardiac tissue and cardiomyocyte samples were immediately fixed overnight at 4°C using TEM fixation solution (G1102, Servicebio).

Techniques: Extraction, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Transfection

Journal: Cell Biology and Toxicology

Article Title: METTL3, m6A modification, and EGR1: interplay affecting myocardial I/R injury outcomes

doi: 10.1007/s10565-024-09937-7

Figure Lengend Snippet: The effect of EGR1 on mitophagy through the regulation of the JAK2/STAT3 pathway. Note: ( A ) Western blot analysis of the expression and quantification of JAK2/STAT3 pathway-related proteins in cardiomyocytes from different treatment groups; ( B ) Schematic diagram showing the treatment of AG490 after shEGR1 transfection in the H/R damage model; ( C ) Western blot analysis of the expression and quantification of JAK2/STAT3 pathway-related proteins in cardiomyocytes from different treatment groups; ( D ) TEM to observe the morphology of cardiomyocyte mitochondria in each group, with a scale bar of 500 nm and arrows indicating mitochondria; ( E ) Representative immunofluorescence images showing the co-localization of GFP-LC3B (green) and mitochondria (MTR-Red, red) in cardiomyocytes from different treatment groups, with a scale bar of 25 μm, and quantification of the number of co-localized spots between GFP-LC3B and mitochondria, with DAPI (blue) indicating the nucleus; ( F ) Representative immunofluorescence images showing the co-localization of MTR-Green (green) and lysosomes (LTR, red) in cardiomyocytes from different treatment groups, with a scale bar of 50 μm, and quantification of the number of co-localized spots between lysosomes and mitochondria in each cell; ( G ) Western blot analysis of the expression and quantification of mitophagy-related proteins in cardiomyocytes from different treatment groups; ( H ) Assessment of cell viability of cardiomyocytes in different treatment groups using the CCK-8 method; ( I ) Measurement of the levels of cTnI and CK-MB in the supernatant of cardiomyocytes in different treatment groups using the ELISA method. * indicates a significant difference between two groups with P < 0.05, ** indicates a significant difference between two groups with P < 0.01, *** indicates a significant difference between two groups with P < 0.001, **** indicates a significant difference between two groups with P < 0.0001. All experiments were repeated three times

Article Snippet: The processed cardiac tissue and cardiomyocyte samples were immediately fixed overnight at 4°C using TEM fixation solution (G1102, Servicebio).

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Biology and Toxicology

Article Title: METTL3, m6A modification, and EGR1: interplay affecting myocardial I/R injury outcomes

doi: 10.1007/s10565-024-09937-7

Figure Lengend Snippet: The effect of METTL3 on the regulation of EGR1 in mitophagy and mitochondrial dynamics. Note: ( A ) Schematic diagram illustrating EGR1-OE or NC-OE transfection; ( B ) Expression of EGR1 in different groups of cardiomyocytes measured by RT-qPCR; ( C ) Schematic diagram illustrating H/R treatment of cardiomyocytes co-transfected with shNC+NC-OE, shMETTL3+NC-OE, or shMETTL3+EGR1-OE; ( D ) TEM images observing the morphology of mitochondria in different groups of cardiomyocytes, Scale bar=500 nm, blue arrows indicate mitochondria; ( E ) Representative immunofluorescence images of cardiomyocytes showing the co-localization of autophagosomes (green, GFP-LC3B) and mitochondria (red, MTR-Red), with quantitative results of GFP-LC3B and mitochondria co-localization spots provided, DAPI (blue, nucleus); Scale bar=25 μm; ( F ) Representative immunofluorescence images of cardiomyocytes showing the co-localization of mitochondria (green, MTR-Green) and lysosomes (red, LTR), with quantitative results of lysosomes and mitochondria co-localization spots provided in each cell; Scale bar=50 μm; ( G ) Expression and quantification of mitophagy-related proteins in different groups of cardiomyocytes measured by Western blot; ( H ) ATP content in different groups of cardiomyocytes; ( I ) Representative images of cardiomyocytes showing MMP detected by JC-1 staining, Scale bar=50 μm, with quantification of the red/green fluorescence ratio provided; ( J ) Expression and quantification of respiratory chain complex-related proteins in different groups of cardiomyocytes measured by Western blot; ( K ) Detection of total ROS and mitochondrial ROS in different groups of cardiomyocytes by DCFDA and MitoSOX staining combined with flow cytometry; ( L ) Concentration of GPX, GSH, and SOD in different groups of cardiomyocytes measured by ELISA; ( M ) Expression and quantification of proteins related to mitochondrial fission and fusion in different groups of cardiomyocytes measured by Western blot; ( N ) Representative immunostaining images of cardiomyocytes showing Tom20 (Scale bar=25 μm) and average length of mitochondria in each cell; ** indicates a significant difference ( p < 0.01) between two groups, *** indicates a highly significant difference ( p < 0.001) between two groups, **** indicates an extremely significant difference ( p < 0.0001) between two groups; all experiments were repeated 3 times

Article Snippet: The processed cardiac tissue and cardiomyocyte samples were immediately fixed overnight at 4°C using TEM fixation solution (G1102, Servicebio).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Immunofluorescence, Western Blot, Staining, Fluorescence, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunostaining