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prolong gold anti fade reagent with dapi  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc prolong gold anti fade reagent with dapi
    GelMa/SCS-MN promotes re-epithelialization and revascularization in diabetic wounds. (a) Photographs of wounds of diabetic mice at different time after treatments. (b) Quantitative analysis of the relative residual wound areas at different times (n = 6). (c) Sagittal sections of day 3 and day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5, green) and <t>DAPI</t> (blue). Scale bar, 200 μm. (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), Itga5 (green) and labeled with 5-ethynyl-2′-deoxyuridine (EdU, gray) (proliferation), cell nucleus for DAPI (blue). Scale bar, 100 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 5). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 5). (g) Sagittal sections of day7 wounds tissue immunolabeled for Endomucin (Emcn, red) and EdU (green), cell nucleus for DAPI (blue). Scale bar, 200 μm. (h) Quantitative statistics of the Emcn + vascular gap in wound (n = 5). (i) Representative H&E staining of diabetic wound tissue on day7 and day 14. The green line indicates epidermal gap. Scale bar, 200 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day 7 and day 14 according to HE staining images (n = 3). (l) Sagittal sections of day14 wounds tissue immunolabeled for Emcn (red), cell nucleus for DAPI (blue). Scale bar, 200 μm. Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA with Tukey's multiple comparison test for (f), (h) and (j), two-way ANOVA with Sidak's multiple comparison test for (b), (e), and (k). P value: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
    Prolong Gold Anti Fade Reagent With Dapi, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+fade+reagent+with+dapi/pmc12547169-303-18-24
    Average 86 stars, based on 1 article reviews
    prolong gold anti fade reagent with dapi - by Bioz Stars, 2026-09
    86/100 stars

    Images

    1) Product Images from "Sulfated polysaccharide facilitates macrophage-Treg crosstalk to mitigate chronic inflammation in diabetic wound healing"

    Article Title: Sulfated polysaccharide facilitates macrophage-Treg crosstalk to mitigate chronic inflammation in diabetic wound healing

    Journal: Bioactive Materials

    doi: 10.1016/j.bioactmat.2025.09.044

    GelMa/SCS-MN promotes re-epithelialization and revascularization in diabetic wounds. (a) Photographs of wounds of diabetic mice at different time after treatments. (b) Quantitative analysis of the relative residual wound areas at different times (n = 6). (c) Sagittal sections of day 3 and day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5, green) and DAPI (blue). Scale bar, 200 μm. (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), Itga5 (green) and labeled with 5-ethynyl-2′-deoxyuridine (EdU, gray) (proliferation), cell nucleus for DAPI (blue). Scale bar, 100 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 5). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 5). (g) Sagittal sections of day7 wounds tissue immunolabeled for Endomucin (Emcn, red) and EdU (green), cell nucleus for DAPI (blue). Scale bar, 200 μm. (h) Quantitative statistics of the Emcn + vascular gap in wound (n = 5). (i) Representative H&E staining of diabetic wound tissue on day7 and day 14. The green line indicates epidermal gap. Scale bar, 200 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day 7 and day 14 according to HE staining images (n = 3). (l) Sagittal sections of day14 wounds tissue immunolabeled for Emcn (red), cell nucleus for DAPI (blue). Scale bar, 200 μm. Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA with Tukey's multiple comparison test for (f), (h) and (j), two-way ANOVA with Sidak's multiple comparison test for (b), (e), and (k). P value: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
    Figure Legend Snippet: GelMa/SCS-MN promotes re-epithelialization and revascularization in diabetic wounds. (a) Photographs of wounds of diabetic mice at different time after treatments. (b) Quantitative analysis of the relative residual wound areas at different times (n = 6). (c) Sagittal sections of day 3 and day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5, green) and DAPI (blue). Scale bar, 200 μm. (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), Itga5 (green) and labeled with 5-ethynyl-2′-deoxyuridine (EdU, gray) (proliferation), cell nucleus for DAPI (blue). Scale bar, 100 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 5). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 5). (g) Sagittal sections of day7 wounds tissue immunolabeled for Endomucin (Emcn, red) and EdU (green), cell nucleus for DAPI (blue). Scale bar, 200 μm. (h) Quantitative statistics of the Emcn + vascular gap in wound (n = 5). (i) Representative H&E staining of diabetic wound tissue on day7 and day 14. The green line indicates epidermal gap. Scale bar, 200 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day 7 and day 14 according to HE staining images (n = 3). (l) Sagittal sections of day14 wounds tissue immunolabeled for Emcn (red), cell nucleus for DAPI (blue). Scale bar, 200 μm. Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA with Tukey's multiple comparison test for (f), (h) and (j), two-way ANOVA with Sidak's multiple comparison test for (b), (e), and (k). P value: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

    Techniques Used: Immunolabeling, Labeling, Migration, Staining, Comparison

    Lacking Tregs impairs SCS-mediated diabetic wound healing. (a) Schematic illustrating experiment depleting Tregs. (b) Flow cytometric quantification of Tregs (Foxp3 + ) in the diabetic skin wound after anti-CD25 mAb injection (n = 6). (c) Representative wound images of diabetic mice after the indicated treatments and the corresponding time-course quantification of relative residual wound area (n = 8). (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5) (green), EdU (gray) and DAPI (blue). Scale bar, 200 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 8). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 12). (g) Representative flow cytometric plots of CD31 + Emcn + cells expression in diabetic wound tissue on day7 post-wounding and corresponding quantitative results (n = 6). (h) Representative immunostainings of CD31 (red), Emcn (green), EdU (gray) and DAPI (blue) in diabetic wound tissue on day7 post-wounding. Scale bar, 200 μm. (i) Representative H&E staining of diabetic wound tissue on day7 and day14. Scale bar, 500 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day14 according to H&E staining images (n = 3). Data are presented as mean ± SD and statistical significance was analyzed via two-way ANOVA with Sidak's multiple comparison test for (c), unpaired two-tailed Student's t -test for (b), (e), (f), (g), (j) and (k). P value: ∗P < 0.05, ∗∗P < 0.01.
    Figure Legend Snippet: Lacking Tregs impairs SCS-mediated diabetic wound healing. (a) Schematic illustrating experiment depleting Tregs. (b) Flow cytometric quantification of Tregs (Foxp3 + ) in the diabetic skin wound after anti-CD25 mAb injection (n = 6). (c) Representative wound images of diabetic mice after the indicated treatments and the corresponding time-course quantification of relative residual wound area (n = 8). (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5) (green), EdU (gray) and DAPI (blue). Scale bar, 200 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 8). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 12). (g) Representative flow cytometric plots of CD31 + Emcn + cells expression in diabetic wound tissue on day7 post-wounding and corresponding quantitative results (n = 6). (h) Representative immunostainings of CD31 (red), Emcn (green), EdU (gray) and DAPI (blue) in diabetic wound tissue on day7 post-wounding. Scale bar, 200 μm. (i) Representative H&E staining of diabetic wound tissue on day7 and day14. Scale bar, 500 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day14 according to H&E staining images (n = 3). Data are presented as mean ± SD and statistical significance was analyzed via two-way ANOVA with Sidak's multiple comparison test for (c), unpaired two-tailed Student's t -test for (b), (e), (f), (g), (j) and (k). P value: ∗P < 0.05, ∗∗P < 0.01.

    Techniques Used: Injection, Immunolabeling, Migration, Expressing, Staining, Comparison, Two Tailed Test

    Related Articles

    Microscopy:

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    Article Snippet: .. The samples were mounted in Anti-Fade Reagent with DAPI (Cell Signaling Technology, Cat. #8961S), and imaged with a confocal microscope (Leica, STELLARIS 5). .. ImageJ software was used to quantify the immunofluorescence signal.

    Article Title: Inhibition of TAF1B impairs ribosome biosynthesis and suppresses cell proliferation in stomach adenocarcinoma through promoting c-MYC mRNA degradation
    Article Snippet: Then, they were permeabilized with 0.5 % PBS-Triton X-100 for 10 min and blocked with 5 % goat serum for 1 h. Subsequently, the slides were incubated overnight with the indicated antibodies at 4 °C, followed by the appropriate secondary antibody, either 488 conjugated goat anti-mouse IgG (ab150120, Abcam, Cambridge, UK) or 594 conjugated goat anti-mouse IgG (ab150077, Abcam, Cambridge, UK). .. The slides were mounted in an Anti-fade Reagent with DAPI (#8961, Cell Signaling Technologies, Danvers, MA, USA) and analyzed using microscopy. .. To conduct the EdU Cell Proliferation Assay, gastric cancer cells were treated with the BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor 488 (C0071S, Beyotime, Shanghai, China) following the manufacturer's instructions.

    Incubation:

    Article Title: Rosuvastatin Attenuates CD40L-Induced Downregulation of Extracellular Matrix Production in Human Aortic Smooth Muscle Cells via TRAF6-JNK-NF-κB Pathway
    Article Snippet: The cells were incubated with secondary goat anti-rabbit IgG-PE conjugate (Cat#sc3739, Santa Cruz; 1:100) or goat anti-mouse IgG-FITC conjugate (Cat#sc2010, Santa Cruz; 1:100). .. The slides were washed 3 times in between each step, and lastly, incubated with anti-fade reagent with DAPI (Cat#8961, CST, USA). .. The cells were observed under a confocal laser scanning microscopy (LSM710, ZEISS, Germany).

    Bicinchoninic Acid Protein Assay:

    Article Title: Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury
    Article Snippet: .. Reagents were as follows: LPS from Escherichia coli O111:B4 (Sigma-Aldrich, #L4391), LPS from Escherichia coli 055:B5 (Sigma-Aldrich, #L2880), human CIRBP/CIRP (Sino Biological, #14578-H07E), rhEPO (Sunshine Pharmaceutical, Shenyang, China), cell dissociation buffer (Gibco, #13150016), PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, #23225), TRIzol Reagent (Sigma-Aldrich, #T9424), cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, #04693159001), GoScriptTM Reverse Transcription System (Promega, #A2800), GoTaq ® qPCR Master Mix (Promega, #A6001), M-PER Protein Extraction Reagent (Thermo Fisher Scientific, #78510), PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616), Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500), LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, #740740), Immunofluorescence Application Solutions Kit (CST, #12727), Anti-fade Reagent with DAPI (Coolaber, #SL 1841), and PE Annexin V Apoptosis Detection Kit (BD, #559763). .. Antibodies were as follows: APC-labeled anti-mouse F4/80 antibody (BioLegend, #123116, clone #BM8), APC/Cyanine7-labeled anti-mouse F4/80 antibody (BioLegend, #123118, clone #BM8), PE-labeled anti-mouse F4/80 antibody (BioLegend, #123110, clone #BM8), PE-labeled anti-mouse Ly-6G antibody (BioLegend, #127608, clone #1A8), APC/Cyanine7-labeled anti-mouse Ly-6G antibody (BioLegend, #127624, clone #1A8), APC/Cyanine7-labeled anti-mouse/human CD11b antibody (BioLegend, #101226, clone #M1/70), APC-labeled anti-mouse TNF-α antibody (BioLegend, #506308, clone #MP6-XT22), APC-labeled anti-mouse IL-6 antibody (BioLegend, #504508, clone #MP5-20F3), APC-labeled anti-mouse IL-10 antibody (BioLegend, #505010, clone #JES5-16E3), PE-labeled anti-mouse EPOR antibody (Bioss, #bs-1424R), FITC-labeled anti-mouse CD86 (BD, #553691, clone #GL-1), APC-labeled anti-mouse CD86 (BioLegend, #105012, clone #GL-1), PE-labeled anti-mouse CD80 (eBioscience, #12-0801-81, clone #16-10A1), PE/Cyanine7-labeled anti-mouse CD80 (BioLegend, #104734, clone #16-10A1), APC-labeled anti-mouse CD206 (BioLegend, #141707, clone #C068C2), PE-labeled anti-mouse CD206 (BioLegend, #141706, clone #C068C2), eBioscienceTM Fixable Viability Dye eFluorTM 450 (Invitrogen, #65-0863-14), anti-CD16/32 antibody (Sungene Biotech, #M10161-14F); Mouse Erythropoietin R Antibody (RD, #AF1390), anti-PPARγ antibody (CTS, #2430), anti-Rab26 antibody (Abcam, #ab198202), anti-β-Actin antibody (CST, #4970S), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-035-003), mouse anti-goat IgG-FITC (Santa Cruz, #sc-2356), and CIRP polyclonal antibody (proteintech, #10209-2-AP).

    Protease Inhibitor:

    Article Title: Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury
    Article Snippet: .. Reagents were as follows: LPS from Escherichia coli O111:B4 (Sigma-Aldrich, #L4391), LPS from Escherichia coli 055:B5 (Sigma-Aldrich, #L2880), human CIRBP/CIRP (Sino Biological, #14578-H07E), rhEPO (Sunshine Pharmaceutical, Shenyang, China), cell dissociation buffer (Gibco, #13150016), PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, #23225), TRIzol Reagent (Sigma-Aldrich, #T9424), cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, #04693159001), GoScriptTM Reverse Transcription System (Promega, #A2800), GoTaq ® qPCR Master Mix (Promega, #A6001), M-PER Protein Extraction Reagent (Thermo Fisher Scientific, #78510), PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616), Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500), LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, #740740), Immunofluorescence Application Solutions Kit (CST, #12727), Anti-fade Reagent with DAPI (Coolaber, #SL 1841), and PE Annexin V Apoptosis Detection Kit (BD, #559763). .. Antibodies were as follows: APC-labeled anti-mouse F4/80 antibody (BioLegend, #123116, clone #BM8), APC/Cyanine7-labeled anti-mouse F4/80 antibody (BioLegend, #123118, clone #BM8), PE-labeled anti-mouse F4/80 antibody (BioLegend, #123110, clone #BM8), PE-labeled anti-mouse Ly-6G antibody (BioLegend, #127608, clone #1A8), APC/Cyanine7-labeled anti-mouse Ly-6G antibody (BioLegend, #127624, clone #1A8), APC/Cyanine7-labeled anti-mouse/human CD11b antibody (BioLegend, #101226, clone #M1/70), APC-labeled anti-mouse TNF-α antibody (BioLegend, #506308, clone #MP6-XT22), APC-labeled anti-mouse IL-6 antibody (BioLegend, #504508, clone #MP5-20F3), APC-labeled anti-mouse IL-10 antibody (BioLegend, #505010, clone #JES5-16E3), PE-labeled anti-mouse EPOR antibody (Bioss, #bs-1424R), FITC-labeled anti-mouse CD86 (BD, #553691, clone #GL-1), APC-labeled anti-mouse CD86 (BioLegend, #105012, clone #GL-1), PE-labeled anti-mouse CD80 (eBioscience, #12-0801-81, clone #16-10A1), PE/Cyanine7-labeled anti-mouse CD80 (BioLegend, #104734, clone #16-10A1), APC-labeled anti-mouse CD206 (BioLegend, #141707, clone #C068C2), PE-labeled anti-mouse CD206 (BioLegend, #141706, clone #C068C2), eBioscienceTM Fixable Viability Dye eFluorTM 450 (Invitrogen, #65-0863-14), anti-CD16/32 antibody (Sungene Biotech, #M10161-14F); Mouse Erythropoietin R Antibody (RD, #AF1390), anti-PPARγ antibody (CTS, #2430), anti-Rab26 antibody (Abcam, #ab198202), anti-β-Actin antibody (CST, #4970S), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-035-003), mouse anti-goat IgG-FITC (Santa Cruz, #sc-2356), and CIRP polyclonal antibody (proteintech, #10209-2-AP).

    Reverse Transcription:

    Article Title: Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury
    Article Snippet: .. Reagents were as follows: LPS from Escherichia coli O111:B4 (Sigma-Aldrich, #L4391), LPS from Escherichia coli 055:B5 (Sigma-Aldrich, #L2880), human CIRBP/CIRP (Sino Biological, #14578-H07E), rhEPO (Sunshine Pharmaceutical, Shenyang, China), cell dissociation buffer (Gibco, #13150016), PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, #23225), TRIzol Reagent (Sigma-Aldrich, #T9424), cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, #04693159001), GoScriptTM Reverse Transcription System (Promega, #A2800), GoTaq ® qPCR Master Mix (Promega, #A6001), M-PER Protein Extraction Reagent (Thermo Fisher Scientific, #78510), PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616), Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500), LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, #740740), Immunofluorescence Application Solutions Kit (CST, #12727), Anti-fade Reagent with DAPI (Coolaber, #SL 1841), and PE Annexin V Apoptosis Detection Kit (BD, #559763). .. Antibodies were as follows: APC-labeled anti-mouse F4/80 antibody (BioLegend, #123116, clone #BM8), APC/Cyanine7-labeled anti-mouse F4/80 antibody (BioLegend, #123118, clone #BM8), PE-labeled anti-mouse F4/80 antibody (BioLegend, #123110, clone #BM8), PE-labeled anti-mouse Ly-6G antibody (BioLegend, #127608, clone #1A8), APC/Cyanine7-labeled anti-mouse Ly-6G antibody (BioLegend, #127624, clone #1A8), APC/Cyanine7-labeled anti-mouse/human CD11b antibody (BioLegend, #101226, clone #M1/70), APC-labeled anti-mouse TNF-α antibody (BioLegend, #506308, clone #MP6-XT22), APC-labeled anti-mouse IL-6 antibody (BioLegend, #504508, clone #MP5-20F3), APC-labeled anti-mouse IL-10 antibody (BioLegend, #505010, clone #JES5-16E3), PE-labeled anti-mouse EPOR antibody (Bioss, #bs-1424R), FITC-labeled anti-mouse CD86 (BD, #553691, clone #GL-1), APC-labeled anti-mouse CD86 (BioLegend, #105012, clone #GL-1), PE-labeled anti-mouse CD80 (eBioscience, #12-0801-81, clone #16-10A1), PE/Cyanine7-labeled anti-mouse CD80 (BioLegend, #104734, clone #16-10A1), APC-labeled anti-mouse CD206 (BioLegend, #141707, clone #C068C2), PE-labeled anti-mouse CD206 (BioLegend, #141706, clone #C068C2), eBioscienceTM Fixable Viability Dye eFluorTM 450 (Invitrogen, #65-0863-14), anti-CD16/32 antibody (Sungene Biotech, #M10161-14F); Mouse Erythropoietin R Antibody (RD, #AF1390), anti-PPARγ antibody (CTS, #2430), anti-Rab26 antibody (Abcam, #ab198202), anti-β-Actin antibody (CST, #4970S), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-035-003), mouse anti-goat IgG-FITC (Santa Cruz, #sc-2356), and CIRP polyclonal antibody (proteintech, #10209-2-AP).

    Real-time Polymerase Chain Reaction:

    Article Title: Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury
    Article Snippet: .. Reagents were as follows: LPS from Escherichia coli O111:B4 (Sigma-Aldrich, #L4391), LPS from Escherichia coli 055:B5 (Sigma-Aldrich, #L2880), human CIRBP/CIRP (Sino Biological, #14578-H07E), rhEPO (Sunshine Pharmaceutical, Shenyang, China), cell dissociation buffer (Gibco, #13150016), PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, #23225), TRIzol Reagent (Sigma-Aldrich, #T9424), cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, #04693159001), GoScriptTM Reverse Transcription System (Promega, #A2800), GoTaq ® qPCR Master Mix (Promega, #A6001), M-PER Protein Extraction Reagent (Thermo Fisher Scientific, #78510), PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616), Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500), LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, #740740), Immunofluorescence Application Solutions Kit (CST, #12727), Anti-fade Reagent with DAPI (Coolaber, #SL 1841), and PE Annexin V Apoptosis Detection Kit (BD, #559763). .. Antibodies were as follows: APC-labeled anti-mouse F4/80 antibody (BioLegend, #123116, clone #BM8), APC/Cyanine7-labeled anti-mouse F4/80 antibody (BioLegend, #123118, clone #BM8), PE-labeled anti-mouse F4/80 antibody (BioLegend, #123110, clone #BM8), PE-labeled anti-mouse Ly-6G antibody (BioLegend, #127608, clone #1A8), APC/Cyanine7-labeled anti-mouse Ly-6G antibody (BioLegend, #127624, clone #1A8), APC/Cyanine7-labeled anti-mouse/human CD11b antibody (BioLegend, #101226, clone #M1/70), APC-labeled anti-mouse TNF-α antibody (BioLegend, #506308, clone #MP6-XT22), APC-labeled anti-mouse IL-6 antibody (BioLegend, #504508, clone #MP5-20F3), APC-labeled anti-mouse IL-10 antibody (BioLegend, #505010, clone #JES5-16E3), PE-labeled anti-mouse EPOR antibody (Bioss, #bs-1424R), FITC-labeled anti-mouse CD86 (BD, #553691, clone #GL-1), APC-labeled anti-mouse CD86 (BioLegend, #105012, clone #GL-1), PE-labeled anti-mouse CD80 (eBioscience, #12-0801-81, clone #16-10A1), PE/Cyanine7-labeled anti-mouse CD80 (BioLegend, #104734, clone #16-10A1), APC-labeled anti-mouse CD206 (BioLegend, #141707, clone #C068C2), PE-labeled anti-mouse CD206 (BioLegend, #141706, clone #C068C2), eBioscienceTM Fixable Viability Dye eFluorTM 450 (Invitrogen, #65-0863-14), anti-CD16/32 antibody (Sungene Biotech, #M10161-14F); Mouse Erythropoietin R Antibody (RD, #AF1390), anti-PPARγ antibody (CTS, #2430), anti-Rab26 antibody (Abcam, #ab198202), anti-β-Actin antibody (CST, #4970S), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-035-003), mouse anti-goat IgG-FITC (Santa Cruz, #sc-2356), and CIRP polyclonal antibody (proteintech, #10209-2-AP).

    Protein Extraction:

    Article Title: Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury
    Article Snippet: .. Reagents were as follows: LPS from Escherichia coli O111:B4 (Sigma-Aldrich, #L4391), LPS from Escherichia coli 055:B5 (Sigma-Aldrich, #L2880), human CIRBP/CIRP (Sino Biological, #14578-H07E), rhEPO (Sunshine Pharmaceutical, Shenyang, China), cell dissociation buffer (Gibco, #13150016), PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, #23225), TRIzol Reagent (Sigma-Aldrich, #T9424), cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, #04693159001), GoScriptTM Reverse Transcription System (Promega, #A2800), GoTaq ® qPCR Master Mix (Promega, #A6001), M-PER Protein Extraction Reagent (Thermo Fisher Scientific, #78510), PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616), Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500), LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, #740740), Immunofluorescence Application Solutions Kit (CST, #12727), Anti-fade Reagent with DAPI (Coolaber, #SL 1841), and PE Annexin V Apoptosis Detection Kit (BD, #559763). .. Antibodies were as follows: APC-labeled anti-mouse F4/80 antibody (BioLegend, #123116, clone #BM8), APC/Cyanine7-labeled anti-mouse F4/80 antibody (BioLegend, #123118, clone #BM8), PE-labeled anti-mouse F4/80 antibody (BioLegend, #123110, clone #BM8), PE-labeled anti-mouse Ly-6G antibody (BioLegend, #127608, clone #1A8), APC/Cyanine7-labeled anti-mouse Ly-6G antibody (BioLegend, #127624, clone #1A8), APC/Cyanine7-labeled anti-mouse/human CD11b antibody (BioLegend, #101226, clone #M1/70), APC-labeled anti-mouse TNF-α antibody (BioLegend, #506308, clone #MP6-XT22), APC-labeled anti-mouse IL-6 antibody (BioLegend, #504508, clone #MP5-20F3), APC-labeled anti-mouse IL-10 antibody (BioLegend, #505010, clone #JES5-16E3), PE-labeled anti-mouse EPOR antibody (Bioss, #bs-1424R), FITC-labeled anti-mouse CD86 (BD, #553691, clone #GL-1), APC-labeled anti-mouse CD86 (BioLegend, #105012, clone #GL-1), PE-labeled anti-mouse CD80 (eBioscience, #12-0801-81, clone #16-10A1), PE/Cyanine7-labeled anti-mouse CD80 (BioLegend, #104734, clone #16-10A1), APC-labeled anti-mouse CD206 (BioLegend, #141707, clone #C068C2), PE-labeled anti-mouse CD206 (BioLegend, #141706, clone #C068C2), eBioscienceTM Fixable Viability Dye eFluorTM 450 (Invitrogen, #65-0863-14), anti-CD16/32 antibody (Sungene Biotech, #M10161-14F); Mouse Erythropoietin R Antibody (RD, #AF1390), anti-PPARγ antibody (CTS, #2430), anti-Rab26 antibody (Abcam, #ab198202), anti-β-Actin antibody (CST, #4970S), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-035-003), mouse anti-goat IgG-FITC (Santa Cruz, #sc-2356), and CIRP polyclonal antibody (proteintech, #10209-2-AP).

    Western Blot:

    Article Title: Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury
    Article Snippet: .. Reagents were as follows: LPS from Escherichia coli O111:B4 (Sigma-Aldrich, #L4391), LPS from Escherichia coli 055:B5 (Sigma-Aldrich, #L2880), human CIRBP/CIRP (Sino Biological, #14578-H07E), rhEPO (Sunshine Pharmaceutical, Shenyang, China), cell dissociation buffer (Gibco, #13150016), PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, #23225), TRIzol Reagent (Sigma-Aldrich, #T9424), cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, #04693159001), GoScriptTM Reverse Transcription System (Promega, #A2800), GoTaq ® qPCR Master Mix (Promega, #A6001), M-PER Protein Extraction Reagent (Thermo Fisher Scientific, #78510), PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616), Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500), LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, #740740), Immunofluorescence Application Solutions Kit (CST, #12727), Anti-fade Reagent with DAPI (Coolaber, #SL 1841), and PE Annexin V Apoptosis Detection Kit (BD, #559763). .. Antibodies were as follows: APC-labeled anti-mouse F4/80 antibody (BioLegend, #123116, clone #BM8), APC/Cyanine7-labeled anti-mouse F4/80 antibody (BioLegend, #123118, clone #BM8), PE-labeled anti-mouse F4/80 antibody (BioLegend, #123110, clone #BM8), PE-labeled anti-mouse Ly-6G antibody (BioLegend, #127608, clone #1A8), APC/Cyanine7-labeled anti-mouse Ly-6G antibody (BioLegend, #127624, clone #1A8), APC/Cyanine7-labeled anti-mouse/human CD11b antibody (BioLegend, #101226, clone #M1/70), APC-labeled anti-mouse TNF-α antibody (BioLegend, #506308, clone #MP6-XT22), APC-labeled anti-mouse IL-6 antibody (BioLegend, #504508, clone #MP5-20F3), APC-labeled anti-mouse IL-10 antibody (BioLegend, #505010, clone #JES5-16E3), PE-labeled anti-mouse EPOR antibody (Bioss, #bs-1424R), FITC-labeled anti-mouse CD86 (BD, #553691, clone #GL-1), APC-labeled anti-mouse CD86 (BioLegend, #105012, clone #GL-1), PE-labeled anti-mouse CD80 (eBioscience, #12-0801-81, clone #16-10A1), PE/Cyanine7-labeled anti-mouse CD80 (BioLegend, #104734, clone #16-10A1), APC-labeled anti-mouse CD206 (BioLegend, #141707, clone #C068C2), PE-labeled anti-mouse CD206 (BioLegend, #141706, clone #C068C2), eBioscienceTM Fixable Viability Dye eFluorTM 450 (Invitrogen, #65-0863-14), anti-CD16/32 antibody (Sungene Biotech, #M10161-14F); Mouse Erythropoietin R Antibody (RD, #AF1390), anti-PPARγ antibody (CTS, #2430), anti-Rab26 antibody (Abcam, #ab198202), anti-β-Actin antibody (CST, #4970S), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-035-003), mouse anti-goat IgG-FITC (Santa Cruz, #sc-2356), and CIRP polyclonal antibody (proteintech, #10209-2-AP).

    Immunofluorescence:

    Article Title: Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury
    Article Snippet: .. Reagents were as follows: LPS from Escherichia coli O111:B4 (Sigma-Aldrich, #L4391), LPS from Escherichia coli 055:B5 (Sigma-Aldrich, #L2880), human CIRBP/CIRP (Sino Biological, #14578-H07E), rhEPO (Sunshine Pharmaceutical, Shenyang, China), cell dissociation buffer (Gibco, #13150016), PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, #23225), TRIzol Reagent (Sigma-Aldrich, #T9424), cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, #04693159001), GoScriptTM Reverse Transcription System (Promega, #A2800), GoTaq ® qPCR Master Mix (Promega, #A6001), M-PER Protein Extraction Reagent (Thermo Fisher Scientific, #78510), PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616), Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500), LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, #740740), Immunofluorescence Application Solutions Kit (CST, #12727), Anti-fade Reagent with DAPI (Coolaber, #SL 1841), and PE Annexin V Apoptosis Detection Kit (BD, #559763). .. Antibodies were as follows: APC-labeled anti-mouse F4/80 antibody (BioLegend, #123116, clone #BM8), APC/Cyanine7-labeled anti-mouse F4/80 antibody (BioLegend, #123118, clone #BM8), PE-labeled anti-mouse F4/80 antibody (BioLegend, #123110, clone #BM8), PE-labeled anti-mouse Ly-6G antibody (BioLegend, #127608, clone #1A8), APC/Cyanine7-labeled anti-mouse Ly-6G antibody (BioLegend, #127624, clone #1A8), APC/Cyanine7-labeled anti-mouse/human CD11b antibody (BioLegend, #101226, clone #M1/70), APC-labeled anti-mouse TNF-α antibody (BioLegend, #506308, clone #MP6-XT22), APC-labeled anti-mouse IL-6 antibody (BioLegend, #504508, clone #MP5-20F3), APC-labeled anti-mouse IL-10 antibody (BioLegend, #505010, clone #JES5-16E3), PE-labeled anti-mouse EPOR antibody (Bioss, #bs-1424R), FITC-labeled anti-mouse CD86 (BD, #553691, clone #GL-1), APC-labeled anti-mouse CD86 (BioLegend, #105012, clone #GL-1), PE-labeled anti-mouse CD80 (eBioscience, #12-0801-81, clone #16-10A1), PE/Cyanine7-labeled anti-mouse CD80 (BioLegend, #104734, clone #16-10A1), APC-labeled anti-mouse CD206 (BioLegend, #141707, clone #C068C2), PE-labeled anti-mouse CD206 (BioLegend, #141706, clone #C068C2), eBioscienceTM Fixable Viability Dye eFluorTM 450 (Invitrogen, #65-0863-14), anti-CD16/32 antibody (Sungene Biotech, #M10161-14F); Mouse Erythropoietin R Antibody (RD, #AF1390), anti-PPARγ antibody (CTS, #2430), anti-Rab26 antibody (Abcam, #ab198202), anti-β-Actin antibody (CST, #4970S), Peroxidase AffiniPure Goat Anti-Rabbit IgG (Jackson Immunoresearch, #111-035-003), mouse anti-goat IgG-FITC (Santa Cruz, #sc-2356), and CIRP polyclonal antibody (proteintech, #10209-2-AP).



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    Image Search Results


    GelMa/SCS-MN promotes re-epithelialization and revascularization in diabetic wounds. (a) Photographs of wounds of diabetic mice at different time after treatments. (b) Quantitative analysis of the relative residual wound areas at different times (n = 6). (c) Sagittal sections of day 3 and day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5, green) and DAPI (blue). Scale bar, 200 μm. (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), Itga5 (green) and labeled with 5-ethynyl-2′-deoxyuridine (EdU, gray) (proliferation), cell nucleus for DAPI (blue). Scale bar, 100 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 5). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 5). (g) Sagittal sections of day7 wounds tissue immunolabeled for Endomucin (Emcn, red) and EdU (green), cell nucleus for DAPI (blue). Scale bar, 200 μm. (h) Quantitative statistics of the Emcn + vascular gap in wound (n = 5). (i) Representative H&E staining of diabetic wound tissue on day7 and day 14. The green line indicates epidermal gap. Scale bar, 200 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day 7 and day 14 according to HE staining images (n = 3). (l) Sagittal sections of day14 wounds tissue immunolabeled for Emcn (red), cell nucleus for DAPI (blue). Scale bar, 200 μm. Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA with Tukey's multiple comparison test for (f), (h) and (j), two-way ANOVA with Sidak's multiple comparison test for (b), (e), and (k). P value: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

    Journal: Bioactive Materials

    Article Title: Sulfated polysaccharide facilitates macrophage-Treg crosstalk to mitigate chronic inflammation in diabetic wound healing

    doi: 10.1016/j.bioactmat.2025.09.044

    Figure Lengend Snippet: GelMa/SCS-MN promotes re-epithelialization and revascularization in diabetic wounds. (a) Photographs of wounds of diabetic mice at different time after treatments. (b) Quantitative analysis of the relative residual wound areas at different times (n = 6). (c) Sagittal sections of day 3 and day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5, green) and DAPI (blue). Scale bar, 200 μm. (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), Itga5 (green) and labeled with 5-ethynyl-2′-deoxyuridine (EdU, gray) (proliferation), cell nucleus for DAPI (blue). Scale bar, 100 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 5). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 5). (g) Sagittal sections of day7 wounds tissue immunolabeled for Endomucin (Emcn, red) and EdU (green), cell nucleus for DAPI (blue). Scale bar, 200 μm. (h) Quantitative statistics of the Emcn + vascular gap in wound (n = 5). (i) Representative H&E staining of diabetic wound tissue on day7 and day 14. The green line indicates epidermal gap. Scale bar, 200 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day 7 and day 14 according to HE staining images (n = 3). (l) Sagittal sections of day14 wounds tissue immunolabeled for Emcn (red), cell nucleus for DAPI (blue). Scale bar, 200 μm. Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA with Tukey's multiple comparison test for (f), (h) and (j), two-way ANOVA with Sidak's multiple comparison test for (b), (e), and (k). P value: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

    Article Snippet: The specimens were sectioned to a thickness of 10 μm, washed three times with TBST, and mounted using ProLong gold anti-fade reagent with DAPI (Cell Signaling Technology).

    Techniques: Immunolabeling, Labeling, Migration, Staining, Comparison

    Lacking Tregs impairs SCS-mediated diabetic wound healing. (a) Schematic illustrating experiment depleting Tregs. (b) Flow cytometric quantification of Tregs (Foxp3 + ) in the diabetic skin wound after anti-CD25 mAb injection (n = 6). (c) Representative wound images of diabetic mice after the indicated treatments and the corresponding time-course quantification of relative residual wound area (n = 8). (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5) (green), EdU (gray) and DAPI (blue). Scale bar, 200 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 8). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 12). (g) Representative flow cytometric plots of CD31 + Emcn + cells expression in diabetic wound tissue on day7 post-wounding and corresponding quantitative results (n = 6). (h) Representative immunostainings of CD31 (red), Emcn (green), EdU (gray) and DAPI (blue) in diabetic wound tissue on day7 post-wounding. Scale bar, 200 μm. (i) Representative H&E staining of diabetic wound tissue on day7 and day14. Scale bar, 500 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day14 according to H&E staining images (n = 3). Data are presented as mean ± SD and statistical significance was analyzed via two-way ANOVA with Sidak's multiple comparison test for (c), unpaired two-tailed Student's t -test for (b), (e), (f), (g), (j) and (k). P value: ∗P < 0.05, ∗∗P < 0.01.

    Journal: Bioactive Materials

    Article Title: Sulfated polysaccharide facilitates macrophage-Treg crosstalk to mitigate chronic inflammation in diabetic wound healing

    doi: 10.1016/j.bioactmat.2025.09.044

    Figure Lengend Snippet: Lacking Tregs impairs SCS-mediated diabetic wound healing. (a) Schematic illustrating experiment depleting Tregs. (b) Flow cytometric quantification of Tregs (Foxp3 + ) in the diabetic skin wound after anti-CD25 mAb injection (n = 6). (c) Representative wound images of diabetic mice after the indicated treatments and the corresponding time-course quantification of relative residual wound area (n = 8). (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5) (green), EdU (gray) and DAPI (blue). Scale bar, 200 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 8). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 12). (g) Representative flow cytometric plots of CD31 + Emcn + cells expression in diabetic wound tissue on day7 post-wounding and corresponding quantitative results (n = 6). (h) Representative immunostainings of CD31 (red), Emcn (green), EdU (gray) and DAPI (blue) in diabetic wound tissue on day7 post-wounding. Scale bar, 200 μm. (i) Representative H&E staining of diabetic wound tissue on day7 and day14. Scale bar, 500 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day14 according to H&E staining images (n = 3). Data are presented as mean ± SD and statistical significance was analyzed via two-way ANOVA with Sidak's multiple comparison test for (c), unpaired two-tailed Student's t -test for (b), (e), (f), (g), (j) and (k). P value: ∗P < 0.05, ∗∗P < 0.01.

    Article Snippet: The specimens were sectioned to a thickness of 10 μm, washed three times with TBST, and mounted using ProLong gold anti-fade reagent with DAPI (Cell Signaling Technology).

    Techniques: Injection, Immunolabeling, Migration, Expressing, Staining, Comparison, Two Tailed Test

    Rh -B8 infection-induced host cell apoptosis in vivo and in vitro . (a) Survival analysis of Ifnar -/- mice infected with Rh -B8 ( n = 9) compared to control mice injected with SPG buffer ( n = 6). All mice died within one week following intravenous injection of Rh -B8. (b) Spleen tissue from Rh -B8-infected Ifnar -/- mice exhibited enlargement and more severe pathological changes compared to uninfected mice. TUNEL staining on splenic paraffin sections revealed a multitude of apoptotic cells (red). Scale bar, 20 µm. Nuclei were indicated by blue DAPI staining. (c) Bacterial counts in tissues, including the spleen, liver, kidney, lung, and brain from five Rh -B8-infected Ifnar -/- mice, were quantified using plaque assay. Means are shown with error bars representing ±SEM. (d) During infection at a multiplicity of infection (MOI) of 1.0, Rh -B8 exhibited slow propagation in the early stages of infection, followed by exponential growth starting at 48 hours post-infection (hpi). In parallel, the viability of HMEC-1 cells decreased significantly by 72 hpi. Data are expressed as mean ± standard deviation (SD), with each experiment conducted in biological triplicates, and results were averaged across all replicates. (e) Immunofluorescence microscopy images demonstrated an increase in the number of apoptotic HMEC-1 cells during Rh -B8 infection. Apoptotic cells were labeled using TUNEL staining (green), nuclei were visualized with DAPI (blue), and bacteria were detected using an anti-OmpB antibody (red). Scale bars, 20 µm.

    Journal: Infection and Immunity

    Article Title: Rickettsia heilongjiangensis suppresses RIPK1 kinase-mediated host cell death during the infection

    doi: 10.1128/iai.00158-25

    Figure Lengend Snippet: Rh -B8 infection-induced host cell apoptosis in vivo and in vitro . (a) Survival analysis of Ifnar -/- mice infected with Rh -B8 ( n = 9) compared to control mice injected with SPG buffer ( n = 6). All mice died within one week following intravenous injection of Rh -B8. (b) Spleen tissue from Rh -B8-infected Ifnar -/- mice exhibited enlargement and more severe pathological changes compared to uninfected mice. TUNEL staining on splenic paraffin sections revealed a multitude of apoptotic cells (red). Scale bar, 20 µm. Nuclei were indicated by blue DAPI staining. (c) Bacterial counts in tissues, including the spleen, liver, kidney, lung, and brain from five Rh -B8-infected Ifnar -/- mice, were quantified using plaque assay. Means are shown with error bars representing ±SEM. (d) During infection at a multiplicity of infection (MOI) of 1.0, Rh -B8 exhibited slow propagation in the early stages of infection, followed by exponential growth starting at 48 hours post-infection (hpi). In parallel, the viability of HMEC-1 cells decreased significantly by 72 hpi. Data are expressed as mean ± standard deviation (SD), with each experiment conducted in biological triplicates, and results were averaged across all replicates. (e) Immunofluorescence microscopy images demonstrated an increase in the number of apoptotic HMEC-1 cells during Rh -B8 infection. Apoptotic cells were labeled using TUNEL staining (green), nuclei were visualized with DAPI (blue), and bacteria were detected using an anti-OmpB antibody (red). Scale bars, 20 µm.

    Article Snippet: Finally, the cell side of the coverslip was inverted onto a microscope slide containing a DAPI anti-fade reagent (Beyotime, P0131).

    Techniques: Infection, In Vivo, In Vitro, Control, Injection, TUNEL Assay, Staining, Plaque Assay, Standard Deviation, Immunofluorescence, Microscopy, Labeling, Bacteria

    Inhibition of caspase-8 does not sensitize Rh -B8-infected HMEC-1 cells to necroptosis. (a and b) Rh -B8-infected cells treated with the caspase-3 inhibitor (Z-DEVD-FMK) showed a significant reduction in cell death during the late stages of infection. An immunofluorescence assay showcased images of TUNEL (green) and DAPI (blue) in HMEC-1 cells infected with Rickettsia (red) at 72 hpi. The growth curve illustrates the replication of Rh -B8 and cell viability of HMEC-1 treated or untreated with Z-DEVD-FMK. (c and d) Rh -B8-infected HMEC-1 cells treated with the caspase-8 inhibitor (Z-IETD-FMK) did not show protection against apoptosis at 72 hpi. The replication of Rh -B8 and cell viability of HMEC-1 between the Z-IETD-FMK-treated and untreated groups exhibited no significant difference. (e and f) The activity of RIPK1 kinase, blocked by necrostatin-1, also did not influence rickettsial growth or host cell viability. (g) The Western blot assay demonstrated that HMEC-1 cells infected with Rh -B8 and treated with the RIPK1 inhibitor necrostatin-1 (Nec-1) at various stages continued to undergo apoptosis. Representative results from three independent experiments are presented for the blotting assays. Uninfected cells treated with each inhibitor for 72 hours were included as controls in panels (a), (c), and (e). Data in (b), (d), and (f) were shown as mean ± SD (each time point has three biological replicates). *** P < 0.001 relative to normal infection, P -values were calculated using an unpaired t -test (two-tailed).

    Journal: Infection and Immunity

    Article Title: Rickettsia heilongjiangensis suppresses RIPK1 kinase-mediated host cell death during the infection

    doi: 10.1128/iai.00158-25

    Figure Lengend Snippet: Inhibition of caspase-8 does not sensitize Rh -B8-infected HMEC-1 cells to necroptosis. (a and b) Rh -B8-infected cells treated with the caspase-3 inhibitor (Z-DEVD-FMK) showed a significant reduction in cell death during the late stages of infection. An immunofluorescence assay showcased images of TUNEL (green) and DAPI (blue) in HMEC-1 cells infected with Rickettsia (red) at 72 hpi. The growth curve illustrates the replication of Rh -B8 and cell viability of HMEC-1 treated or untreated with Z-DEVD-FMK. (c and d) Rh -B8-infected HMEC-1 cells treated with the caspase-8 inhibitor (Z-IETD-FMK) did not show protection against apoptosis at 72 hpi. The replication of Rh -B8 and cell viability of HMEC-1 between the Z-IETD-FMK-treated and untreated groups exhibited no significant difference. (e and f) The activity of RIPK1 kinase, blocked by necrostatin-1, also did not influence rickettsial growth or host cell viability. (g) The Western blot assay demonstrated that HMEC-1 cells infected with Rh -B8 and treated with the RIPK1 inhibitor necrostatin-1 (Nec-1) at various stages continued to undergo apoptosis. Representative results from three independent experiments are presented for the blotting assays. Uninfected cells treated with each inhibitor for 72 hours were included as controls in panels (a), (c), and (e). Data in (b), (d), and (f) were shown as mean ± SD (each time point has three biological replicates). *** P < 0.001 relative to normal infection, P -values were calculated using an unpaired t -test (two-tailed).

    Article Snippet: Finally, the cell side of the coverslip was inverted onto a microscope slide containing a DAPI anti-fade reagent (Beyotime, P0131).

    Techniques: Inhibition, Infection, Immunofluorescence, TUNEL Assay, Activity Assay, Western Blot, Two Tailed Test