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

Journal: Molecular Metabolism

Article Title: Loss of Tbk1 kinase activity protects mice from diet-induced metabolic dysfunction

doi: 10.1016/j.molmet.2018.06.007

Figure Lengend Snippet: Tbk1 Δ/ Δ mice have less inflammation relative to Tbk1 +/ + mice on HFD . (A) Representative 20x images and quantification of cd11b/c stained subcutaneous white adipose tissue from Tbk1 Δ/Δ and Tbk1 +/+ mice after 10 weeks on ND or HFD (n = 4–8 mice/group). Scale bar indicates 200 μM and ‘CLS’ refers to crown like structures. (B) mRNA expression of genes encoding CD11c, F4/80, TNFα, IL-6 and IL-12 in subcutaneous WAT of Tbk1 Δ/Δ and Tbk1 +/+ mice fed with HFD as indicated (n = 4–6 mice/group). (C) Liver tissue lysates from 14-week-old HFD-fed Tbk1 Δ/Δ and Tbk1 +/+ mice were immunoblotted with antibodies against IL-6 and IL-1β. β-actin and GAPDH were used as internal loading controls. Indicated cytokines from WAT (D) and liver tissue (E) lysates of HFD-fed Tbk1 +/+ and Tbk1 Δ/Δ mice were measured by Bio-Rad multiplex array (n = 4–8 mice/group for WAT, n = 8–12 mice/group for liver). All mice are from 129S5 background. Results are representative of mean +/− SEM. Statistical analysis by Student's t -test. * p < 0.05, ** p < 0.01.

Article Snippet: Sections for immunohistochemical analysis were blocked with 20% aquablock and incubated with rabbit anti-cd11b/c (Novus Biologicals, NB110-40766) in blocking solution (5% BSA in TBS with 0.05% tween) at 4 °C overnight.

Techniques: Staining, Expressing, Multiplex Assay

Journal: Frontiers in Oncology

Article Title: Prognostic stratification based on the levels of tumor-infiltrating myeloid-derived suppressor cells and PD-1/PD-L1 axis in locally advanced rectal cancer

doi: 10.3389/fonc.2022.1018700

Figure Lengend Snippet: Representative immunofluorescence images of two cases with (A−D) high (upper row) and (E−H) low (lower row) number of tumor-infiltrating MDSCs. Based on the expression of (A, E) CD33, (B, F) CD11b, and (C, G) DAPI, (D, H) the merged triple immunofluorescence results were assessed. Cells with yellow color were merged from red and green. yellow arrows: CD11b + CD33 - cells; white arrows: CD11b + CD33 + cells; Scale bar: 20 μm. MDSC, myeloid-derived suppressor cell; DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: Subsequently, nonspecific antigens were blocked using 0.2% bovine serum albumin in PBS for 1 h. For the double-staining of CD11b and CD33, a cocktail of primary antibodies including anti-CD11b (#NB110-89474, Novus Biologics; 1:800) and anti-CD33 (#MAB11371-100, R&D systems; 1:500), was incubated in blocking solution overnight at 4°C.

Techniques: Immunofluorescence, Expressing, Derivative Assay