rabbit anti human a 2b  (Alomone Labs)

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A ) Immunohistochemical (IHC) staining to detect platelets (CD41 + ) in healthy liver biopsies (Normal) and those from patients with AILI (Patient). Scale bar, 250 μm (n = 10/group). ( B ) Male C57B/6 mice treated with PBS or acetaminophen (APAP). Intravital microscopy analyses were performed around 3 hr post-APAP. Mɸs (cyan) and platelets (white) in liver sinusoids (red) are indicated. Representative images were chosen from intravital microscopy videos: https://bcm.box.com/s/15hmtryyrdl302mihrsm034ure87x4ea (Supplementary video 1, PBS treatment) and https://bcm.box.com/s/tuljfmstvv4lvoksx16fkxkpirkekynz (Supplementary video 2; n = 6–7 mice/group, 4–15 videos/mouse). ( C–E ) Male C57B/6 (wild-type [WT]) mice were treated with control IgG (Ctrl IgG) or an anti-CD41 antibody (α-CD41 Ab) either 3 hr before or 3 hr after APAP administration. ( C ) Serum levels of ALT and ( D ) liver histology with necrotic areas outlined were evaluated 24 hr after APAP treatment (n = 5 mice/group in C , D ). Scale bar, 250 μm. ( E ) Male C57B/6 (WT) and Chil1 -/- mice were treated with APAP. Additionally, Chil1 -/- mice were divided into two groups treated with either PBS or recombinant mouse Chi3l1 (rmChi3l1) simultaneously with APAP. Immunofluorescence (IF) staining was performed to detect intrahepatic platelets (CD41 + ) 3 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 μm. Two-tailed, unpaired Student’s t-test was performed in A–C . One-way ANOVA were performed in E .

Article Snippet: Antibody , Rabbit polyclonal anti-human CD41 , Proteintech , 24552–1-AP, RRID: AB_2879604 , 1:200 for IHC.

Techniques: Immunohistochemical staining, Immunohistochemistry, Intravital Microscopy, Control, Recombinant, Immunofluorescence, Staining, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: Male C57B/6 mice were treated with control IgG (Ctrl IgG) or an anti-CD41 antibody (α-CD41 Ab) either 3 hr before (pre-) or 3 hr after (post-) APAP administration. Immunofluorescence (IF) staining was performed to identify intrahepatic platelets (CD41 + ) (n = 5 mice/group). Scale bar, 25 μm.

Article Snippet: Antibody , Rabbit polyclonal anti-human CD41 , Proteintech , 24552–1-AP, RRID: AB_2879604 , 1:200 for IHC.

Techniques: Control, Immunofluorescence, Staining

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A ) Immunoprecipitation with anti-CD44 antibody was performed using liver homogenates obtained from wild-type (WT) and Cd44 -/- mice treated with acetaminophen (APAP) for 2 hr. Input proteins and immune-precipitated proteins were blotted with the indicated antibodies. ( B ) Interferometry measurement of the binding kinetics of human His-Chi3l1 with human Fc-CD44. ( C ) His-tagged control GFP and human Chi3l1 were incubated with recombinant human CD44. Proteins bound to Chi3l1 were immune-precipitated with an anti-His antibody. Input proteins and immune-precipitated proteins were blotted with indicated antibodies. ( D–F ) Male WT mice were treated with APAP and Cd44 -/- mice were treated with PBS or recombinant mouse Chi3l1 (rmChi3l1) plus APAP. ( D ) Serum levels of ALT and ( E ) liver histology with necrotic areas outlined were evaluated 24 hr after APAP treatment (n = 4–9 mice/group in A , B ). Scale bar, 250 μm. ( F ) Immunofluorescence (IF) staining was performed to detect intrahepatic platelets (CD41 + ) 3 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 μm. One-way ANOVA were performed in D .

Article Snippet: Antibody , Rabbit polyclonal anti-human CD41 , Proteintech , 24552–1-AP, RRID: AB_2879604 , 1:200 for IHC.

Techniques: Immunoprecipitation, Binding Assay, Control, Incubation, Recombinant, Immunofluorescence, Staining

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A–C ) Chil1 -/- mice reconstituted with recombinant mouse Chi3l1 (rmChi3l1) were treated with either Ctrl IgG or α-CD44 Ab 30 min prior to acetaminophen (APAP) challenge. ( A ) Serum levels of ALT and ( B ) liver histology with necrotic areas outlined were evaluated 24 hr after APAP treatment (n = 4–5 mice/group). Scale bar, 250 μm. ( C ) Immunofluorescence (IF) staining was performed to detect intrahepatic platelets (CD41 + ) 3 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 μm. ( D ) Flow cytometry analysis was performed to identify Chi3l1-binding cells among liver non-parenchymal cells (NPCs) isolated from wild-type (WT) mice treated with APAP for 2 hr. CD44 + cells were gated from single live cells. CD44 + cells that bind to rmChi3l1 were further gated. The Chi3l1 + CD44 + cells were then identified by markers for various cell types, including CD45 + CD146 - F4/80 + (Mɸs), CD45 - CD146 + (liver sinusoidal endothelial cells [LSECs]), and Ly6G + (neutrophils). Two-tailed, unpaired Student’s t-test was performed in A .

Article Snippet: Antibody , Rabbit polyclonal anti-human CD41 , Proteintech , 24552–1-AP, RRID: AB_2879604 , 1:200 for IHC.

Techniques: Recombinant, Immunofluorescence, Staining, Flow Cytometry, Binding Assay, Isolation, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A ) Immunohistochemical (IHC) staining for macrophages (CD68 + ) and platelets (CD41 + ) in normal liver biopsies (Normal) and those from patients with AILI (Patient) (n = 10/group). Scale bar, 25 μm. ( B ) Immunofluorescence (IF) staining for intrahepatic platelets (CD41 + ) and Kupffer cells (KCs) (F4/80 + ) in male C57B/6 mice treated with PBS or acetaminophen (APAP) for 3 hr. Scale bar, 25 μm. Arrowheads indicate platelets adherent to KCs. Quantification of platelets adherent to KCs or liver sinusoidal endothelial cells (LSECs). ( C–F ) Male C57B/6 mice were injected with either empty liposomes containing PBS (PBS) or liposomes containing clodronate (CLDN), followed by APAP treatment. ( C, D ) Non-parenchymal cells (NPCs) were isolated and underwent flow cytometry analysis. Indicated cells were gated on single live CD45 + CD146 - cells. IF staining was performed to detect intrahepatic platelets (CD41 + ). Scale bar, 25 μm. ( E ) Serum levels of ALT and ( F ) liver histology with necrotic areas outlined. Scale bar, 250 μm (n = 6 mice/group in B-F ). Two-tailed, unpaired Student’s t-test was performed in B-D , F .

Article Snippet: Antibody , Rabbit polyclonal anti-human CD41 , Proteintech , 24552–1-AP, RRID: AB_2879604 , 1:200 for IHC.

Techniques: Immunohistochemical staining, Immunohistochemistry, Immunofluorescence, Staining, Injection, Liposomes, Isolation, Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A ) Male WT, Chil1 -/- , Cd44 -/- mice were treated with acetaminophen (APAP) (n = 4 mice/group). After 3 hr, mice were sacrificed and Mɸs were isolated to measure mRNA levels of various adhesion molecules, including selectin P ligand ( Selplg ), Cd40 , melanoma cell adhesion molecule ( Mcam ), Fc receptor ( Fcr ), intercellular adhesion molecule 1 ( Icam1 ), lymphocyte function-associated antigen 1 ( Lfa1 ), von Willebrand factor ( Vwf ), and podoplanin ( Pdpn ). ( B, C ) Wild-type (WT) mice were treated with APAP. Chil1 -/- and Cd44 -/- mice were treated with PBS or rmChi3l1 followed by APAP challenge simultaneously and mice were sacrificed 3 hr after APAP (n = 3 mice/group). ( B ) Mɸs were isolated and mRNA levels of Pdpn in Mɸs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). ( C ) Immunofluorescence (IF) staining of liver sections for podoplanin and F4/80 is shown and the proportions of Mɸs that express Pdpn were quantified, Scale bar, 25 μm. ( D–F ) Chil1 -/- mice reconstituted with rmChi3l1 were treated with either Ctrl IgG or α-podoplanin Ab for 16 hr and subsequently challenged with APAP. ( D ) Serum levels of ALT and ( E ) liver histology were evaluated 24 hr after APAP treatment (n = 6 mice/group). Scale bar, 250 μm. ( F ) IF staining for intrahepatic platelets (CD41 + ) and Mɸs (F4/80+) was performed 3 hr after APAP (n = 3 mice/group). Scale bar, 25 μm. One-way ANOVA were performed in A–C . Two-tailed, unpaired Student’s t-test was performed in D .

Article Snippet: Antibody , Rabbit polyclonal anti-human CD41 , Proteintech , 24552–1-AP, RRID: AB_2879604 , 1:200 for IHC.

Techniques: Isolation, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Staining, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet: ( A–C ) Male C57B/6 mice were treated with acetaminophen (APAP) for 3 hr, followed by intraperitoneally ( i.p. ) injection of either a control IgG (Ctrl IgG) or an anti-mouse Chi3l1 Ab (α-mChi3l1 Ab, C59). ( A ) Immunofluorescence (IF) staining for intrahepatic platelets (CD41 + ) was performed 6 hr after APAP treatment (n = 3 mice/group). Scale bar, 25 μm. ( B ) Serum levels of ALT and ( C ) liver histology were evaluated 24 hr after APAP treatment (n = 4–6 mice/group). Scale bar, 250 μm. ( D–F ) Chil1 -/- mice were treated with APAP plus PBS or recombinant human Chi3l1 (rhChi3l1) for 3 hr as indicated and APAP plus rhChi3l1 treatment group were either without treatment or treated with a control IgG (Ctrl IgG) or an anti-human Chi3l1 Ab (α-hChi3l1 Ab, C7). ( D ) IF staining was performed to identify intrahepatic platelets (CD41 + ) 6 hr after APAP treatment. Scale bar, 25 μm. ( E ) Serum levels of ALT and ( F ) liver histology were evaluated 24 hr after APAP treatment. Scale bar, 250 μm (n = 5–10 mice/group in D–F ). Two-tailed, unpaired Student’s t-test was performed in B . One-way ANOVA were performed in E .

Article Snippet: Antibody , Rabbit polyclonal anti-human CD41 , Proteintech , 24552–1-AP, RRID: AB_2879604 , 1:200 for IHC.

Techniques: Injection, Control, Immunofluorescence, Staining, Recombinant, Two Tailed Test

Journal: eLife

Article Title: Chitinase 3-like-1 contributes to acetaminophen-induced liver injury by promoting hepatic platelet recruitment

doi: 10.7554/eLife.68571

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-human CD41 , Proteintech , 24552–1-AP, RRID: AB_2879604 , 1:200 for IHC.

Techniques: Recombinant, Diagnostic Assay, Intravital Microscopy

Journal: Cells

Article Title: Glial A 2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity

doi: 10.3390/cells9051245

Figure Lengend Snippet: Schematic representation of the design of experiments on cultured enteric glial cells (EGCs). EGCs were treated for three days with palmitate (PA, 400 μM). Then, cells were incubated for 2 h with lipopolysaccharide (LPS, 10 μg/mL) before the addition of adenosine deaminase (ADA, 3 U/mL), to minimize interferences by extracellular adenosine. Under these conditions, cells were treated for 19 h with MRS1754 (0.1 µM, A 2B R antagonist) or incubated for 1 h with MRS1754 before the addition of BAY60-6583 (0.05 µM, selective A 2B R agonist, 18 h). For the experiments with the inhibitor of glial cell-derived neurotrophic factor (GDNF) receptor signaling, cells were treated with RPI-1 (100 µM, 1,3-dihydro-5,6-dimethoxy-3-[(4-hydroxyphenyl)methylene]-H-indol-2-one) for 19 h. On the fourth day, cells were lysed for analysis of toll-like receptor 4 (TLR4) expression and the culture media were collected for analysis of substance P (SP), GDNF, and interleukin (IL)-1β release.

Article Snippet: The LMMP preparations were then incubated overnight at room temperature with guinea pig polyclonal anti-rat S100β (1:100; Synaptic Systems, Göttingen, Germany), rabbit polyclonal anti-human A 2B R (1:50; Merck Life Science, Milan, Italy), and mouse biotin-conjugated anti-human-HuC/D (1:100; Thermo Fisher Scientific, Milan, Italy) primary antibodies.

Techniques: Cell Culture, Incubation, Derivative Assay, Expressing

Journal: Cells

Article Title: Glial A 2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity

doi: 10.3390/cells9051245

Figure Lengend Snippet: ( A ) Representative confocal microphotographs showing the distribution of the neural marker HuC/D (yellow), the glial protein S100β (green), and A 2B Rs (magenta) in colonic longitudinal smooth muscle with attached myenteric plexus (LMMP) whole mount preparations from mice fed with standard diet (SD) or high-fat diet (HFD), in the absence or presence of 50 μM fluorocitrate (FC). White arrowheads indicate HuC/D + and A 2B R + neurons, while white stars indicate S100β + and A 2B R + glial cells. Scale bars = 22 μm; analysis of A 2B R ( B ) and S100B ( C ) density index in colonic LMMP whole mount preparations from mice fed with SD or HFD, in the absence or presence of 50 μM FC. * p < 0.05, ** p < 0.01 versus SD without FC; aa p < 0.01 versus HFD; n = 5 mice per group.

Article Snippet: The LMMP preparations were then incubated overnight at room temperature with guinea pig polyclonal anti-rat S100β (1:100; Synaptic Systems, Göttingen, Germany), rabbit polyclonal anti-human A 2B R (1:50; Merck Life Science, Milan, Italy), and mouse biotin-conjugated anti-human-HuC/D (1:100; Thermo Fisher Scientific, Milan, Italy) primary antibodies.

Techniques: Marker

Journal: Cells

Article Title: Glial A 2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity

doi: 10.3390/cells9051245

Figure Lengend Snippet: Schematic representation of the proposed role of A 2B Rs in the modulation of enteric glial cell (EGC) activity under experimental conditions mimicking obesity. In vitro incubation of EGCs with palmitate (PA, 400 μM) and lipopolysaccharide (LPS, 10 μg/mL) elicited an increase in TLR4 expression as well as substance P (SP), glial cell-derived neurotrophic factor (GDNF), and interleukin (IL)-1β release, suggesting an involvement of these cells in supporting the enteric inflammation and abnormal tachykininergic enteric motor responses associated with obesity. The pharmacological stimulation of A 2B Rs on EGCs, besides reducing TLR4 expression and IL-1β levels, can also counteract the abnormal increase in GDNF and SP release. A 2B R: adenosine A 2B receptor; AC: adenylyl cyclase; ATP: adenosine triphosphate; cAMP: cyclic adenosine monophosphate; NF-ҡB: nuclear factor kappa-light-chain-enhancer of activated B cells.

Article Snippet: The LMMP preparations were then incubated overnight at room temperature with guinea pig polyclonal anti-rat S100β (1:100; Synaptic Systems, Göttingen, Germany), rabbit polyclonal anti-human A 2B R (1:50; Merck Life Science, Milan, Italy), and mouse biotin-conjugated anti-human-HuC/D (1:100; Thermo Fisher Scientific, Milan, Italy) primary antibodies.

Techniques: Activity Assay, In Vitro, Incubation, Expressing, Derivative Assay