fluoromount g with dapi  (SouthernBiotech)

Journal: The Journal of Experimental Medicine

Article Title: Central neurons encode interleukin-1β signals and mediate stress-induced inflammation

doi: 10.1084/jem.20252000

Figure Lengend Snippet: IL-1β induces inflammatory responses in C57BL/6J mice. (A) Serum IL-6 level at 2 h after injection of PBS or IL-1β (PBS, n = 5 mice; IL-1β, n = 5 mice). Unpaired t test. (B) Core body temperature for 6 h after injection of PBS or IL-1β (5 μg/kg). (C) Minimum change in core body temperature at 1 h after PBS or IL-1β injection (PBS, n = 7 mice; IL-1β, n = 8 mice). Data are represented as individual mouse data points pooled from two independent experiments. Unpaired t test. (D) ΔHR for 60 min after PBS or IL-1β injection. (E) Area under the curve (AUC) of change in heart rate (ΔHR) after PBS or IL-1β injection (PBS, n = 3 mice; IL-1β, n = 6 mice). Data are represented as individual mouse data points. This experiment was completed once. Unpaired t test. (F) Serum corticosterone levels at 2 h after PBS or IL-1β injection (PBS, n = 3 mice; IL-1β, n = 3 mice). Data are represented as individual mouse data points. This experiment was completed once. (G) Representative images showing immunofluorescence (IF) staining of the labeled cells (tdTomato, red) co-stained with DAPI (blue) in the NTS and PVN. Scale bar, 200 μm for the NTS. Scale bar, 100 μm for the PVN. (H) Serum IL-6 level at 2 h after injection of saline or CNO in naïve TRAP2-Cre mice (saline, n = 3 mice; CNO, n = 6 mice). Data are represented as individual mouse data points. This experiment was completed once. (I) ΔHR for 60 min after saline or CNO injection in naïve TRAP2-Cre mice (saline, n = 3 mice; CNO, n = 3 mice). *P < 0.05; **P < 0.01. NTS, nucleus tractus solitarius.

Article Snippet: Sections were mounted using Fluoromount-G with DAPI (Southern Biotech) and imaged with BZ-X810 All-in-One Microscope (Keyence) and confocal microscope (Zeiss LSM880; Zeiss), with subsequent analysis performed via FIJI software.

Techniques: Injection, Immunofluorescence, Staining, Labeling, Saline

Journal: The Journal of Experimental Medicine

Article Title: Central neurons encode interleukin-1β signals and mediate stress-induced inflammation

doi: 10.1084/jem.20252000

Figure Lengend Snippet: Distinct neuronal populations in the BNST recapitulate IL-1β responses. (A) STPT of the whole mouse brain of TRAP2-tdTomato mice. Top lane: overview of the STPT data acquisition and registration; middle lane: representative coronal sections; and bottom lane: areas showing tdTomato + neuronal cell bodies. (B) tdTomato + cell count in indicated brain region in IL-1β (5 μg/kg; n = 6) and PBS-injected groups ( n = 6). PVT, paraventricular nucleus of the thalamus; PBNc, parabronchial nucleus complex; LC, locus coeruleus; AP, area postrema; NTS: nucleus of the tractus solitarius. Data are represented as individual mouse data points with mean ± SEM. This experiment was completed once. One-way ANOVA with Tukey’s multiple comparison test. (C) Representative images showing immunofluorescence staining of the labeled cells (tdTomato, red) co-stained with DAPI (blue) in the BNST. tdTomato + cell count in the dorsal and ventral BNST regions after exposure to IL-1β. Data are represented as individual mouse data points pooled from two independent experiments. Unpaired t test. Scale bar, 100 μm. (D) Schematic of TRAPing IL-1β–responsive BNST neurons and their reactivation in TRAP2 mice. (E) Representative images of BNST showing Gq-DREADD-mCherry–expressing cells (red) co-stained with c-Fos (green) after CNO administration. Scale bar, 20 μm. (F) Core body temperature for 6 h after reactivation with saline (black) as a control or CNO (red). (G) Minimum change in core body temperature at 1 h after injection. Data are represented as individual mouse data points pooled from two independent experiments. Unpaired t test. (H) ΔHR for 60 min after reactivation of IL-1β–TRAPed neurons with saline (black) or CNO (red). Saline, n = 8 mice; CNO, n = 10 mice, mixed-effects analysis with Šidák correction. (I) AUC of ΔHR after reactivation. Data are represented as individual mouse data points from three independent experiments. Unpaired t test. (J and K) Serum levels of IL-6 and corticosterone at 2 h after saline or CNO administration. Data are represented as individual mouse data points pooled from three independent experiments. Unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Box and whisker plots show the minimum, maximum, median, and 25th and 75th percentiles.

Article Snippet: Sections were mounted using Fluoromount-G with DAPI (Southern Biotech) and imaged with BZ-X810 All-in-One Microscope (Keyence) and confocal microscope (Zeiss LSM880; Zeiss), with subsequent analysis performed via FIJI software.

Techniques: Cell Characterization, Injection, Comparison, Immunofluorescence, Staining, Labeling, Expressing, Saline, Control, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: Central neurons encode interleukin-1β signals and mediate stress-induced inflammation

doi: 10.1084/jem.20252000

Figure Lengend Snippet: snRNA-seq of BNST cells. (A) A UMAP plot of BNST cells with 17 cell clusters identified by snRNA-seq. (B) UMAP plots showing expression of indicated genes. The clusters showing high expression in are marked in red. (C) Representative images showing CRH expression in IL-1β–responsive BNST neurons: CRH (green), IL-1β–responsive BNST neurons (tdTomato, red), and DAPI (blue). Right panel shows the percentage of tdTomato + CRH + double-positive cells in tdTomato + cells in the BNST. Scale bar, 10 μm.

Article Snippet: Sections were mounted using Fluoromount-G with DAPI (Southern Biotech) and imaged with BZ-X810 All-in-One Microscope (Keyence) and confocal microscope (Zeiss LSM880; Zeiss), with subsequent analysis performed via FIJI software.

Techniques: Expressing

Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: Ferritinophagy aggravated MV-induced pulmonary fibrosis through regulating ferroptosis. (A, B). Immunoblot and quantitative analysis showing the expression levels of ferritinophagy markers NCOA4 and FTH in MLE-12 cells with and without MS. n = 3 per group. (C, D). Immunoblot and quantitative analysis showing the expression levels of NCOA4 and FTH in the lung homogenates. n = 4–6 per group. (E, F). Immunoblot and quantitative analysis showing the expression levels of SLC7A11 and GPX4 in the lung homogenates. n = 4–6 per group. (G, H). Immunoblot and quantitative analysis showing the expression levels of SLC7A11, GPX4 and FTH in DFO-treated MLE-12 cells with and without MS. n = 3 per group. Representative immunofluorescence staining images and relevant quantitative analysis for the epithelial marker E-cadherin (Green) and NCOA4 (Red) (I), or FTH (Green) and NCOA4 (Red) in the lung tissues (J). White arrows point to the colocalization of the two markers. Nuclei was stained by DAPI (blue). Scale bars correspond to 20 μm. MFI, mean fluorescence intensity. (K, L). Immunoblot and quantitative analysis showing the expression levels of fibronectin and α-SMA in the lung homogenates. n = 4–6 per group. (M, N). Representative histopathologic images of H&E and Masson’s staining comparing the lung injury and collagen deposition. Original magnification x 200. Scale bars correspond to 100 μm. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Marker, Fluorescence

Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: Activation of ANG II/AGTR1 pathway initiated NCOA4-mediated ferritinophagy to aggravate MV-induced pulmonary fibrosis. (A, B). Immunoblot and quantitative analysis showing the expression levels of ANG II and AGTR1 in the lung homogenates. n = 4–6 per group. (C, D). Immunoblot and quantitative analysis showing the expression levels of NCOA4 and FTH in the lung homogenates. n = 4–6 per group. (E, F). Immunoblot and quantitative analysis showing the expression levels of SLC7A11 and GPX4 in the lung homogenates. n = 4–6 per group. Representative immunofluorescence staining images and relevant quantitative analysis for the epithelial marker E-cadherin (Green) and NCOA4 (Red) (G), or FTH (Green) and NCOA4 (Red) in the lung tissues (H). White arrows point to the colocalization of the two markers. Nuclei was stained by DAPI (blue). Scale bars correspond to 20 μm. MFI, mean fluorescence intensity. (I, J) Immunoblot and quantitative analysis showing the expression levels of fibronectin and α-SMA in the lung homogenates. n = 4–6 per group. (K, L). Representative histopathologic images of H&E and Masson’s staining comparing the lung injury and collagen deposition. Original magnification x 200. Scale bars correspond to 100 μm. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, Staining, Marker, Fluorescence

Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: Suppression of NCOA4 inhibited ferroptosis and alleviated MV-induced pulmonary fibrosis. (A, B). Immunoblot and quantitative analysis showing the expression levels of NCOA4 and FTH in the lung homogenates. n = 4–6 per group. (C, D). Immunoblot and quantitative analysis showing the expression levels of SLC7A11 and GPX4 in the lung homogenates. n = 4–6 per group. Representative immunofluorescence staining images and relevant quantitative analysis for the epithelial marker E-cadherin (Green) and NCOA4 (Red) (E), or FTH (Green) and NCOA4 (Red) (F) in the lung tissues. White arrows point to the colocalization of the two markers. Nuclei was stained by DAPI (blue). Scale bars correspond to 20 μm. MFI, mean fluorescence intensity. (G, H). Immunoblot and quantitative analysis showing the expression levels of fibronectin and α-SMA in the lung homogenates. n = 4–6 per group. (I, J). Representative histopathologic images of H&E and Masson’s staining comparing the lung injury and collagen deposition. Original magnification x 200. Scale bars correspond to 100 μm. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Marker, Fluorescence

Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: Mechanical ventilation enriched EVs to promote activation of lung fibroblast through iron overload. (A). GO enrichment analysis of differential expressed genes in the type II alveolar epithelial cells and fibroblasts originated from the MV group versus the control group. (B). Immunoblot analysis of the characteristic markers, Alix and CD63, and ferritin of EVs from control-BALF and MV-BALF. n = 3 per group. (C). NTA analysis of the concentration and size distribution of EVs from control-BALF and MV-BALF. (D). TEM images of EVs from control-BALF and MV-BALF. Original magnification x 80,000. Scale bars correspond to 200 nm. (E). Heatmap of the correlation strength of cellular interactions between different cells. (F). Cellchat analysis showing the intracellular interaction strength between AT2 or fibroblasts and other cell types in the MV group. (G, H). Immunoblot and quantitative analysis of fibronectin, α-SMA and FTH in MRC-5 cells treated with EVs. n = 3 per group. (I). Representative images of ferrous iron (red) in the MRC-5 cells treated with PKH-67 labelled EVs (green) by the confocal microscopy. Nuclei was stained by DAPI (blue). Scale bars correspond to 10 μm, and n = 3 biologically independent samples. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Activation Assay, Control, Western Blot, Concentration Assay, Confocal Microscopy, Staining

Journal: Journal of Advanced Research

Article Title: NCOA4-Mediated Ferritinophagy Induces Ferroptosis and Enriches Ferritin-Containing EVs via Ferritin Phase Separation to Promote Mechanical Ventilation-Induced Pulmonary Fibrosis

doi: 10.1016/j.jare.2025.07.043

Figure Lengend Snippet: MV induced ferritinophagy promoted the transfer of iron from EVs to activate lung fibroblast. (A, B). Immunoblot and quantitative analysis of fibronectin, α-SMA and FTH in MRC-5 cells treated with BALF-EVs generated from chloroquine intervened mice. n = 3 per group. (C). Representative images of ferrous iron (red) in the MRC-5 cells treated with PKH-67 labelled EVs (green) by the confocal microscopy. Nuclei was stained by DAPI (blue). (D). Immunofluorescence of α-SMA (red) in MRC-5 cells treated with BALF-EVs generated from chloroquine intervened mice. Nuclei was stained by DAPI (blue). (E, F). Immunoblot and quantitative analysis of fibronectin, α-SMA and FTH in MRC-5 cells treated with BALF-EVs generated from NCOA4-knockdown mice administrated by AAV. n = 3 per group. (G). Representative images of ferrous iron (red) in the MRC-5 cells treated with PKH-67 labelled EVs (green) by the confocal microscopy. Nuclei was stained by DAPI (blue). (H). Immunofluorescence of α-SMA (red) in MRC-5 cells treated with BALF-EVs generated from NCOA4-knockdown mice administrated by AAV. Nuclei was stained by DAPI (blue). Scale bars correspond to 10 μm, and n = 3 biologically independent samples. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The slides were then washed and mounted using DAPI Fluoromount-G (0100–20, Southern Biotechnology, USA).

Techniques: Western Blot, Generated, Confocal Microscopy, Staining, Immunofluorescence, Knockdown