Journal: Journal of Respiration

Article Title: Immune Correlates of Non-Necrotic and Necrotic Granulomas in Pulmonary Tuberculosis: A Pilot Study

doi: 10.3390/jor1040023

Figure Lengend Snippet: Figure 2. Distribution of CD15, S100A8/A9, CD4 and IBA1 positive immune cells in human lung TB granulomas. (A) Representative images singleplex (CD15 and S100A8/A9; red spots) and multiplex (CD4; green spots and IBA1; red spots) immunostaining of control, solid/non-necrotic and necrotic granulomas. Host cell nucleus is stained blue with DAPI. Scale bar in S100A8/A9 panel (50 microns) is common for CD15 and S100A8/A9 images. Scale bar in the IBA1 panel (50 microns) is common for CD4 and IBA1 images. (B) Distribution of CD15 positive cells (including MDSCs) relative to the total number of cells in the field in control, solid/non-necrotic granulomas and necrotic granulomas. (C) Distribution of S100A8/A9 positive cells (mainly neutrophils) relative to the total number of cells in the field in control, solid/non-necrotic granulomas and necrotic granulomas. (D) Distribution of CD4 positive T-cells relative to the total number of cells in the field in control, solid/non-necrotic granulomas and necrotic granulomas. (E) Distribution of IBA1 positive cells (activated macrophages) relative to the total number of cells in the field in control, solid/non-necrotic granulomas and necrotic granulomas. Data plotted in (B–E) are mean and standard error mean (SEM) of n = 3 (control); n = 5 (solid lesion) and n = 18 (necrotic lesion). Data were analyzed by One-way ANOVA with Tukey’s post-hoc correction for multiple group comparison; * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: S100A8/A9) (cat no. ab22506) were purchased from (Abcam, Waltham, MA, USA), CD33 (cat no. sc19660), CD15 (cat no. sc-19649), ARG1 and NOS2 (cat no. sc-7271) (Santa Cruz Biotechnology, Dallas, TX, USA), HIF-1α (cat no. MA5160048) Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), IL-6 (cat no. MAB206) and IL-10 (cat no. AF-217-NA) (R& D Systems Inc, Minneapolis, MN, USA) and CD4 from (cat no. NBP1-19371AF647) Novus Biologicals, Centennial, CO, USA).

Techniques: Multiplex Assay, Immunostaining, Control, Staining, Comparison

Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: Immunostaining after Recording ATP-elicited Ca 2+ Signals in pancreatic macrophages (PMs). (A). Representative images of pancreatic lobule loaded with Fluo-4AM before (Ai) and after ATP (10 µM) application (Aii), the arrow indicates the position of the ( n = 8). A corresponding Fluo 4 trace from a PM is shown in Aiii . Corresponding immunostaining of this lobule with antibodies F4/80 Alexa Fluo 647 is shown below ( Aiv) . Hoechst 33342 staining of the same area is shown in Av . Arrow points to ear-like shape of PM nucleus. Overlay of antibody and Hoechst 33342 staining is shown in Avi . Scale bar is 10µm. (B). Immunostaining of another area in a pancreatic lobule with monoclonal F4/80 antibodies labeled with Alexa Fluor 647 (Bi) . Staining of nuclei in the same lobule with Hoechst 33342 (Bii) . Overlay of B i with Bii is shown in Biii . Scale bar is 10µm. (C). Representative images of a pancreatic lobule loaded with Fluo-4AM before (Ci) and after ATP (10 µM) application (Cii) , the arrow indicates the position of the PM. Corresponding Fluo 4 trace is shown in Ciii. Immunostaining of the same area with monoclonal CD11b antibody conjugated with Alexa Fluor 647 ( n = 8) is shown in Civ . Overlay of Cii and Civ is shown in Cv . Scale bar is 10µm.

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Immunostaining, Staining, Labeling

Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: IgG-elicited Ca 2+ Spikes in PMs . (A). Single short Ca 2+ spike occurring after application of IgG (0.1–0.25 mg/mL) in a PM from a control pancreatic lobule. This was an infrequent observation (5 out of 29 cells tested) and is most likely not an IgG-elicited Ca 2+ signal as such single spikes have been also observed in 3 out of 15 cells in the absence of IgG stimulation. (B) . Representative trace of IgG (0.1–0.25 mg/mL)-induced Ca 2+ signals in PMs in pancreatic lobules isolated from mice with AP (FAEE-AP model—48 h). Such oscillations were observed in 9 out of 31 cells. Single short spikes have been observed in 4 out of 31 cells. No oscillations were observed in the absence of stimulation with IgG ( n = 14), while single short spikes have been observed in 2 out of 14 cells. (C). Average Ca 2+ spike frequencies in PMs displaying Ca 2+ signals under the conditions indicated. The frequencies in control PMs, both stimulated with IgG (blue bar) and unstimulated (green), as well as in unstimulated PMs from the FAEE-AP model (48 h, orange bar) were much lower than in PMs from the FAEE-AP model stimulated with IgG (red bar, P < 0.007). (D) . Average Ca 2+ spike duration in PMs displaying Ca 2+ signals under the conditions indicated. Although the average spike duration was longer in the PMs from the FAEE-AP mice stimulated with IgG than under the other conditions, the difference was not statistically different ( P > 0.2). (E). Representative images of immunostaining of PMs in lobules using antibodies F4/80 conjugated with Alexa Fluor 647. Lobules were isolated from control and FAEE-AP 3-day mice (72 h in vivo FAEE-AP model). Scale bar is 20µm. (F). Comparison of the average density of PMs in lobules from control and FAEE-AP 2-day and 3-day mice (48 h and 72 h in vivo FAEE-AP model, respectively). Control, 2.36 ± 0.6 SEM, n = 14; FAEE-AP 2 day, 9.56 ± 1.86 SEM, * P < 0.033, n = 16; FAEE-AP 3 days, 15.37 ± 1.51 SEM, * P < 0.038 as compared to FAEE-AP 2-day, n = 35. The difference between control and FAEE-AP 3-day was very highly significant (**** P < 0.0001).

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Control, Isolation, Immunostaining, In Vivo, Comparison