Journal: Materials today. Bio

Article Title: Precise delivery of doxorubicin and imiquimod through pH-responsive tumor microenvironment-active targeting micelles for chemo- and immunotherapy.

doi: 10.1016/j.mtbio.2022.100482

Figure Lengend Snippet: Fig. 10. Immunostaining of tumor tissues in tumor-bearing mice after treatments. (A) Immunohistochemistry images of tumor sections stained with CD3, CD8, and TNF-α antibodies. The scale bar is 100 μm. (B) Immunofluorescence images of tumor tissues after treatments for 12 days. The scale bar is 50 μm. Blue fluorescence represents the cell nucleus stained with DAPI. Green fluorescence represents the iNOS stained with the iNOS antibody conjugated with FITC.

Article Snippet: After 30 min, the tissue slice was stained with diluted iNOS antibody (Miltenyi Biotec, catalog: 130-116-357) at 4 C overnight.

Techniques: Immunostaining, Immunohistochemistry, Staining

Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: Mechanisms of IL‐17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats

doi: 10.1111/1440-1681.70116

Figure Lengend Snippet: Effects of IL‐17A neutralisation on macrophage polarisation in SHR renal tissues. (A) Representative IHC staining of M1 macrophage markers (iNOS and CD86) with quantitative analysis of their positive areas. (B) Representative IHC staining of M2 macrophage markers (Arg‐1 and CD163) with quantitative analysis of their positive areas. (C) Representative immunoblots and relative expression levels of iNOS, CD86, Arg‐1, and CD163 proteins. (D) Proportion of CD86 + and CD163 + cells among CD68 + macrophages. (E) mRNA expression levels of iNOS, CD86, Arg‐1, and CD163. Data are presented as mean ± SD ( n = 6).

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA).

Techniques: Immunohistochemistry, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs

doi: 10.1371/journal.pone.0258427

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: NANOG , REA314 , PE , Recombinant human IgG1 , anti-human , 130-117-526 , Miltenyi Biotec.

Techniques: Cytometry, Conjugation Assay, Recombinant, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Acetyl‐11‐keto‐beta‐boswellic acid modulates macrophage polarization and Schwann cell migration to accelerate spinal cord injury repair in rats

doi: 10.1111/cns.14642

Figure Lengend Snippet: Information of primary antibodies.

Article Snippet: INOS , 1:200 , Rabbit , bs‐0162R , AB_10855051 , Bioss (Bei‐jing, China).

Techniques: Concentration Assay

Journal: International journal of molecular medicine

Article Title: Fasudil, a Rho-associated protein kinase inhibitor, attenuates retinal ischemia and reperfusion injury in rats.

doi: 10.3892/ijmm.2011.659

Figure Lengend Snippet: Figure 5. Immunohistochemical staining of iNOS in rat retinas at 1 day after I/R. (A) sham group; (B) I/R group; (C) control group; (D) fasudil group. The rats in I/R group and control group exhibited a remarkable increase in iNOS reactivity (n=5).

Article Snippet: A rabbit polyclonal antibody against rat iNOS (1:100, Boster Bio-Engineering co. Ltd., Wuhan, china) was used as a primary antibody.

Techniques: Immunohistochemical staining, Staining, Control

Journal: International journal of molecular medicine

Article Title: Fasudil, a Rho-associated protein kinase inhibitor, attenuates retinal ischemia and reperfusion injury in rats.

doi: 10.3892/ijmm.2011.659

Figure Lengend Snippet: Figure 6. iNOS mRNA and protein levels in rat retinas at 1 day after I/R were measured by quantitative real-time RT-PCR (A) and Western blot analysis (B), respectively. (A) Compared to the sham group, there was a marked increase in iNOS mRNA in the I/R group and the control group. Fasudil pre-treatment significantly attenuated the increase of iNOS mRNA in retinas of rats with I/R. (B) iNOS protein levels in the above-mentioned groups were consistent with the mRNA expression by Western blotting. Representative blots are shown, and the protein size is expressed in kDa. (n=5, *P<0.05 vs. the sham group, #P<0.01 vs. the control group).

Article Snippet: A rabbit polyclonal antibody against rat iNOS (1:100, Boster Bio-Engineering co. Ltd., Wuhan, china) was used as a primary antibody.

Techniques: Quantitative RT-PCR, Western Blot, Control, Expressing

Journal: Autophagy

Article Title: Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes

doi: 10.1080/15548627.2023.2167689

Figure Lengend Snippet: Inhibition of autophagy is associated with increased expression of pro-inflammatory markers after TBI. (A) Images (20X, scale bar: 50 μm) of Cx3cr1-GFP cortical sections stained with antibodies against pro-inflammatory cytokine NOS2 (red) and autophagy flux marker SQSTM1(purple). (B) Quantification of IF data from (A) measuring NOS2 expression in all CX3CR1 + cells (black bars) and CX3CR1 + cells with inhibited autophagy (gray bars) in sham, and 1-, 3- and 7 days post TBI cortical sections. 69% of all CX3CR1 + NOS2 + cells show inhibition of autophagy at 3 days post injury. (C) Images (20X, scale bar: 50 μm) of Cx3cr1-GFP mouse cortical sections stained with antibodies against inflammasome marker NLRP3 (red) and autophagy flux marker SQSTM1(purple). (D) Quantification of IF data from (C) measuring NLRP3 expression in all CX3CR1 + cells (black bars) and CX3CR1 + cells with inhibited autophagy (gray bars) in sham and 1-, 3- and 7 days post TBI brain cortical sections. 90% of all CX3CR1 + NLRP3 + cells show inhibition of autophagy at 3 days post injury. Data are mean ± SEM, n = 5 mice/group; *p < 0.05, **p < 0.01 vs. corresponding sham (one-way ANOVA with Dunnet’s post-hoc for multiple comparisons). (E-J) Flow cytometry-based assessment of inflammatory markers in microglia and infiltrating myeloid cells with normal autophagy flux (Cyto-ID® autophagy dye − , black) versus inhibited autophagy flux (dye + , red) isolated form sham and injured mouse brains at 1-, 3-, 7- and 28- days post TBI. (E) Representative histograms showing comparison of IL1B/IL-1β staining intensity in microglia with normal autophagy flux (black, dye + ) and inhibited autophagy flux (red, dye − ). (F-G) Quantification of IL1B (F) and TNF/TNF-α (G) MFI in microglia demonstrates increased pro-inflammatory expression levels in cells with inhibited autophagy up to day 28 post injury. (H) Representative histograms showing comparison of IL1B staining intensity in infiltrating myeloid cells with normal (black) and inhibited autophagy flux. (I-J) Quantification of IL1B (I) and TNF (J) mean fluorescence intensity (MFI) in infiltrating myeloid cells. Data are mean ± SEM, n = 6–7 mice/group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. corresponding autophagy dye − group; two-way ANOVA with Bonferroni’s post-hoc for multiple comparisons.

Article Snippet: Primary antibodies used include: CYBB/NOX2 (1:1000; BD Biosciences, 611,414), NLRP3 (1:200; Cell Signaling Technology, 15,101), NOS2/iNOS (1:250; BD Biosciences), AIF1/IBA1 (1:1000; Wako, 019–19,741), SQSTM1/p62 (1:200; Progen, GP62-C), ADGRE1/F4/80 (1:500; Thermo Fisher Scientific, MA1-91,124), and TMEM119 (1:200, Cell Signaling Technology, 90,840).

Techniques: Inhibition, Expressing, Staining, Marker, Flow Cytometry, Isolation, Fluorescence

Journal: Autophagy

Article Title: Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes

doi: 10.1080/15548627.2023.2167689

Figure Lengend Snippet: Inhibition of autophagy in macrophages and microglia exacerbates inflammatory responses in vitro . (A) Western blot of murine IMG microglial cells treated with autophagy inhibitors bafilomycin A 1 (BafA, 20 nM), 3-methyladenine (3-MA, 100 μM), or MRT68921 (MRT, 10 μM) for 6 h with or without lipopolysaccharide (LPS) pre-treatment (10 ng/μl, 3 h). (B) Densitometric analysis from (A) demonstrating increased NOS2 and NLRP3 protein expression (proteins normalized to loading control ACTB/β-Actin) in IMG microglial cells treated with autophagy inhibitors, under both basal conditions and following LPS pre-treatment. (C) Griess assay demonstrating increased nitric oxide production in IMG microglial cells treated with autophagy inhibitors as described in (A). (D) Western blot of murine RAW 246.7 macrophage cells treated with autophagy inhibitors as described in (A). (E) Densitometric analysis from (D) demonstrating increased NOS2 and NLRP3 protein expression in RAW cells treated with autophagy inhibitors, under both basal conditions and following LPS pre-treatment. (F) Griess assay demonstrating increased nitric oxide production in RAW macrophage cells treated with autophagy inhibitors as described in (A). (G) Densitometric analysis of LC3-II and SQSTM1 protein levels from (A) and (D) indicating that inflammation (LPS treatment) itself does not lead to inhibition of autophagy flux in IMG and RAW cells. (H) Western blot of C57Bl/6 bone marrow derived macrophages (BMDM) showing increased expression of NOS2 protein levels when treated with autophagy inhibitors as described in (A). Data are mean ± SEM; n = 3 replicates/group with 3 independent experiments performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, vs corresponding control; one-way ANOVA with Tukey’s post-hoc for multiple comparisons.

Article Snippet: Primary antibodies used include: CYBB/NOX2 (1:1000; BD Biosciences, 611,414), NLRP3 (1:200; Cell Signaling Technology, 15,101), NOS2/iNOS (1:250; BD Biosciences), AIF1/IBA1 (1:1000; Wako, 019–19,741), SQSTM1/p62 (1:200; Progen, GP62-C), ADGRE1/F4/80 (1:500; Thermo Fisher Scientific, MA1-91,124), and TMEM119 (1:200, Cell Signaling Technology, 90,840).

Techniques: Inhibition, In Vitro, Western Blot, Expressing, Griess Assay, Derivative Assay

Journal: Autophagy

Article Title: Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes

doi: 10.1080/15548627.2023.2167689

Figure Lengend Snippet: Microglia and monocyte-specific inhibition of autophagy exacerbates inflammatory responses after TBI. (A) Quantification of microglia and infiltrating monocytes that are tdTomato + , indicative of Lyz2-Cre expression. (B-G) Results of NanoString analysis comparing neuroinflammatory gene expression in cortical tissue from sham and TBI (3 days post injury) control ( Lyz2-cre ) mice and mice with microglia and macrophage-specific inhibition of autophagy ( Lyz2-cre/Becn1-flox , abbreviated as becn1 cKO ). (B) Partial Least Squares – Discriminate Analysis (PLS-DA) plot demonstrating separation among control sham (purple), becn1 cKO sham, control TBI (blue), and becn1 cKO TBI (green) mice groups; R2 = 0.98, Q2 = 0.81. Each point represents a data set from an individual animal. The 95% confidence intervals are indicated by elliptical shaded areas. Data were sum normalized, log transformed, and mean centered. (C) Heatmap including all assessed genes based on t-test/ANOVA, Euclidean distancing, and ward clustering. (B-C) were generated using MetaboAnalyst. (D) Volcano plot highlighting differentially expressed genes between becn1 cKO and control mice in sham (top) and TBI (bottom) cortices. Genes with p < 0.05 and fold change > 2 are highlighted in red. (E) Pathways analysis using NIH-DAVID indicates that innate immune responses are the most differentially regulated between becn1 cKO and control mice in the injured cortex after TBI. (F) Nanostring based heatmap shows increased expression innate immunity genes in the injured cortex of becn1 cKO compared to control mice at 3 days post injury. Color coding was based on z-score scaling. (G) Nanostring based heatmap shows decrease in autophagy gene expression in the injured cortices of becn1 cKO mice compared to control mice at 3 days post injury. Color coding was based on z-score scaling. (H) Western blot of cortical tissue lysates demonstrating that becn1 cKO mice have increased impairments in autophagy compared to control mice after TBI (3 days post injury). (I) Densitometric analysis from (H) shows increased expression of autophagy proteins LC3-II and SQSTM1 in becn1 cKO as compared to control mice at 3 days post injury. (J) qRT-PCR demonstrating that pro-inflammatory genes Cybb and Nfkb1 are significantly higher in the injured cortices of becn1 cKO mice compared to control mice at 3 days post injury. (K) qRT-PCR demonstrating that anti-inflammatory genes Il10 and Tgfb levels are not significantly changed in the injured cortices of control and becn1 cKO mice at 3 days post injury. Data are mean ± SEM; n = 5–6 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001 vs corresponding control; ^^^p < 0.001, ^^^^p < 0.0001 vs corresponding sham (two-way ANOVA with Tukey’s post-hoc for multiple comparisons). (L) Images (20X, scale bar: 50 μm) of control and becn1 cKO cortical sections at 3 days post TBI, stained with antibodies against immune cell marker AIF1/IBA1 (green), pro-inflammatory marker NOS2 (red), and autophagy flux marker SQSTM1 (purple). Corresponding quantification of microglial/macrophage cells with inhibited autophagy (AIF1 + SQSTM1 + ) and increased NOS2 expression (AIF1 + SQSTM1 + NOS2 + ). Data are presented as mean ± SEM; n = 4–5 mice/group. *p < 0.05 vs control (AIF1 + SQSTM1 + cells, Student’s t-test).

Article Snippet: Primary antibodies used include: CYBB/NOX2 (1:1000; BD Biosciences, 611,414), NLRP3 (1:200; Cell Signaling Technology, 15,101), NOS2/iNOS (1:250; BD Biosciences), AIF1/IBA1 (1:1000; Wako, 019–19,741), SQSTM1/p62 (1:200; Progen, GP62-C), ADGRE1/F4/80 (1:500; Thermo Fisher Scientific, MA1-91,124), and TMEM119 (1:200, Cell Signaling Technology, 90,840).

Techniques: Inhibition, Expressing, Transformation Assay, Generated, Western Blot, Quantitative RT-PCR, Staining, Marker