Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Alterations in Gut Microbiome-Host Relationships After Immune Perturbation in Patients With Multiple Sclerosis

doi: 10.1212/NXI.0000000000200355

Figure Lengend Snippet: Flow cytometry was used to determine the proportions of (A) IgA-coated and (B) IgM-coated bacteria. (C) Total secreted, unbound IgA was quantified using ELISA. Multiple linear regression models, adjusted for age, sex, BMI, and steroid use, were applied in (A–C) to assess the significance of differences between controls and patients with untreated MS. BMI = body mass index; IgA = immunoglobulin A; MS = multiple sclerosis.

Article Snippet: Soluble, unbound IgA in the fecal supernatants was quantified using an ELISA kit (Eagle Biosciences, SGA35-K01).

Techniques: Flow Cytometry, Bacteria, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Larval ascariasis elicits a prominent IgA and IgG1/2 antibody response to adult Ascaris excretory/secretory antigens in pigs

doi: 10.3389/fimmu.2025.1606128

Figure Lengend Snippet: Strong positive correlation between sIgA against A . suum adult ES and eosinophil influx. (A) Pearson correlation of sIgA against adult ES products and %eosinophil frequencies of leukocytes in BAL . (B) Box plots illustrating median IgM, IgG and IgA responses against A . suum adult ES products in BAL fluid. (C) Box plots illustrating sIgA against L3 lysate, adult lysate and adult ES in BAL fluid. Blue and red denotes uninfected controls and A. suum infected pigs, respectively. Whiskers indicate 95% percentile. Significance determined by Wilcoxon test is represented by p<0.05: *p<0.01: **.

Article Snippet: For IgM, IgA, IgG1 and IgG2, non-conjugated mouse anti-pig IgM (Bio-Rad, # MCA637GA) diluted at 1:20000, mouse anti-pig IgA (Bio-Rad, # MCA638GA) diluted at 1:1000, mouse anti-pig IgG1 (Bio-Rad, # MCA635GA) diluted at 1:1000 and mouse anti-pig IgG2 (Bio-Rad, # MCA638GA) diluted at 1:1000, mouse anti-pig sIgA (Bio-Rad, # MCA634GA) diluted at 1:10000 was used.

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Larval ascariasis elicits a prominent IgA and IgG1/2 antibody response to adult Ascaris excretory/secretory antigens in pigs

doi: 10.3389/fimmu.2025.1606128

Figure Lengend Snippet: IgA + B cells are highly induced in the ileal mLN 14 dpi. (A) Boxplots of sIgA against the three antigens in intestinal mucus. Exemplary flow cytometry plots depicting the gating strategy used to identify CD79a + B cell populations in mLNs. Representative flow cytometry plots of IgA + cells gated on CD79a + cells and bar plots of mean frequencies of % IgA producing B cells in jejunal (B) and ileal (C) mLN 14 dpi. (D) Representative flow cytometry plots of IgA + cells gated on CD79a + cells and bar plots of mean frequencies of % IgA producing B cells in jejunal mLN 35 dpi. Bar plot error bars indicate mean sd. Boxplot whiskers indicate 95% percentile. Significance determined by Wilcoxon test is represented by p<0.05: *p<0.01: **.

Article Snippet: For IgM, IgA, IgG1 and IgG2, non-conjugated mouse anti-pig IgM (Bio-Rad, # MCA637GA) diluted at 1:20000, mouse anti-pig IgA (Bio-Rad, # MCA638GA) diluted at 1:1000, mouse anti-pig IgG1 (Bio-Rad, # MCA635GA) diluted at 1:1000 and mouse anti-pig IgG2 (Bio-Rad, # MCA638GA) diluted at 1:1000, mouse anti-pig sIgA (Bio-Rad, # MCA634GA) diluted at 1:10000 was used.

Techniques: Flow Cytometry

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: Representative image of leptin expression in lung adenocarcinoma. Leptin expression in tumors and paired normal lung tissues was detected by immunochemical staining analysis. (A) Leptin protein was overexpressed in a moderately differentiated lung adenocarcinoma samples with acinar predominant growth compared with normal control lung tissue. It can be seen that leptin protein was mainly expressed in the parenchyma (epithelial cells) of lung cancer tissues. (B) There was no difference in the expression of leptin protein in a case of well-differentiated lung adenocarcinoma with lepidic predominant (LPA) growth compared with normal control lung tissue. Leptin was seen to be expressed at low levels in the parenchyma of lung cancer tissue and in normal lung tissue. (C) Leptin protein was overexpressed in a highly differentiated lung adenocarcinoma sample with papillary predominant growth (PPA) compared to normal control lung tissue. It can be seen that leptin protein was mainly expressed in the parenchyma of lung cancer tissues. (D) Leptin protein was overexpressed in a poorly differentiated lung adenocarcinoma sample with micropapillary predominant growth (MPA) compared to normal control lung tissue. It can be seen that leptin protein was expressed in both parenchyma and mesenchyme of lung cancer tissues. (E) Leptin protein was overexpressed in a highly differentiated lung adenocarcinoma with solid predominant growth compared to normal control lung tissue. It can be seen that leptin protein was expressed in the parenchyma, mesenchyme, and normal control lung tissues of lung cancer, but the expression level was low in normal tissues. (F) Leptin protein was overexpressed in a lung minimally invasive adenocarcinoma (MIA) compared to normal control lung tissue from the same patient. All experiments were repeated three times. Immunohistochemistry staining IHC staining of leptin was performed according to the manufacturer’s instructions. A streptavidin-peroxidase staining kit was purchased from ZSGB BIO (Beijing, China). The paraffin-embedded tissues were prepared by a pathology specialist, and dewaxed and rehydrated in the lab. The experimental steps were carried out according to the instructions of the SP kit. The tissues were incubated with primary antibodies diluted to the recommended concentration overnight at 4 °C with antibodies that was dilute to the recommended concentration. Then 3,3'-diaminobenzidine (DAB) staining was performed, and the results were observed under a microscope.

Article Snippet: A ntibodys and reagents Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Staining, Immunohistochemistry, Incubation, Concentration Assay, Microscopy

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: The effect of endogenous leptin expression on the proliferation of pulmonary adenocarcinoma cells. (A) The clone formation test of H1299 and A549 cells on the effect of proliferation of pulmonary adenocarcinoma; 50 ng/mL artificial recombinant leptin protein was used to simulate the impact of exogenous leptin in circulating blood on tumor cells; (B) the flow cell cycle analysis of PI staining showing the effect of endogenous leptin expression on the cell cycle of lung adenocarcinoma in H1299 and A549 cells; (C) AV/PI double-staining flow cytometry apoptotic cell test showing the effect of endogenous leptin expression in the apoptosis of lung adenocarcinoma in H1299-sh and A549 cells. All the experiments were repeated three times. Immunohistochemistry staining IHC staining of leptin was performed according to the manufacturer’s instructions. A streptavidin–peroxidase staining kit was purchased from ZSGB BIO (Beijing, China). The paraffin-embedded tissues were prepared by a pathology specialist, and dewaxed and rehydrated in the lab. The experimental steps were carried out according to the instructions of the SP kit. The tissues were incubated with primary antibodies diluted to the recommended concentration overnight at 4 °C with antibodies that was dilute to the recommended concentration. Then 3,3'-diaminobenzidine (DAB) staining was performed, and the results were observed under a microscope (100×).

Article Snippet: A ntibodys and reagents Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Recombinant, Cell Cycle Assay, Staining, Double Staining, Flow Cytometry, Immunohistochemistry, Incubation, Concentration Assay, Microscopy

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: Molecular mechanisms of endogenous leptin expression effect on the proliferative ability of lung adenocarcinoma cells. (A) Effect of endogenous leptin expression on key signaling molecules of PI3K/AKT pathway and its downstream signaling pathway in H1299 and A549 cells detected by western blotting; (B) effect of endogenous leptin expression on the p53 signaling pathway in A549 cells by western blotting; (C) endogenous leptin expression level on the expression level of autophagy-related protein LC3-II in H1299 cell lines detected by protein immunofluorescence. (D) Effect of endogenous leptin expression on key signaling molecules of the mTOR pathway and its downstream signalings in H1299 and A549 cells detected by western blotting. All the experiments were repeated three times. Scale bar: 50 µm.

Article Snippet: A ntibodys and reagents Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Immunofluorescence