Intestinal tissues were collected and fixed overnight in 4% paraformaldehyde. The fixed intestinal tissues were OCT-embedded, snap-frozen, and prepared into 16 μm sections for immunofluorescence staining. Cell samples including the primary EGCs, primary astrocytes and Caco-2 cells were seeded in 24-well plates plated with cell climbing slices respectively. After stimulation, cell samples were fixed in 4% paraformaldehyde and washed thrice with PBS for immunofluorescence staining. After permeabilization and blocking, the tissue sections or cell samples were incubated with the respective primary antibodies overnight at 4 °C. Briefly, the primary antibodies consisted of chicken anti-GFAP (1:200, GeneTex, Irvine, CA, USA), rabbit anti-Iba-1 (1:200 Abcam, Cambridge, UK), rabbit anti-Sox10 (1:500, Abcam, Cambridge, UK), anti-CD40 (1:200, Abcam, Cambridge, UK), and ZO-1 (1:200, Abcam, Cambridge, UK) antibodies. Next, the tissue sections or cell samples were washed thrice with PBS and incubated with A488 anti-chicken (1:1000, GeneTex) or A594 anti-rabbit (1:1000, GeneTex) antibodies at 37 °C for 2 h. DAPI (1:1000, Invitrogen) was used for nuclei counterstaining. An Olympus FluoView FV1000 microscope (Olympus, Tokyo, Japan) was used to collect the fluorescent images of intestinal tissues and cell samples. Fluorescent intensity was analyzed by ImageJ.
Anti cd40
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-CD40 is a laboratory reagent used for research purposes. It is an antibody that binds to the CD40 protein, which is expressed on the surface of various cell types, including B cells, dendritic cells, and some tumor cells. The primary function of Anti-CD40 is to enable researchers to study the role of CD40 in cellular processes and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview fv1000 microscope, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Rabbit anti iba1, by Abcam (2 mentions)
Rabbit anti sox10, by Abcam (2 mentions)
Chicken anti gfap, by GeneTex (2 mentions)
A488 anti chicken, by GeneTex (2 mentions)
A594 anti rabbit, by GeneTex (2 mentions)
45 ti rotor, by Beckman Coulter (1 mentions)
Jem 1011 electron microscope, by JEOL (1 mentions)
Quixstand, by GE Healthcare (1 mentions)
0.22 μm bottle top filter, by Merck Group (1 mentions)
Anti cd81, by Abcam (1 mentions)
Fitc anti mouse cd19, by BioLegend (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti icos, by Abcam (1 mentions)
Fitc anti mouse cd3ε, by BioLegend (1 mentions)
Anti icosl, by Abcam (1 mentions)
Anti cd4, by Santa Cruz Biotechnology (1 mentions)
Anti cd40l, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
7 protocols using anti cd40
1
Immunofluorescence Staining of Intestinal Tissues and Cells
2
Isolation and Characterization of Adipose Stem Cell-Derived Extracellular Vesicles
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As in our previous study [11 (link), 24 (link), 25 ], adipose tissue was obtained from the abdominal fat of C57BL/6 mice. ASCs were cultured at 37°C with 5% CO2 in α-modified Eagle's medium (α-MEM) containing 10% fetal bovine serum (FBS) until 1 × 106 cells/cm2 were obtained. EVs were isolated from ASC sup as previously described [26 (link)]. The supernatant was filtered through a 0.45 μm vacuum filter. The filtrate was concentrated using QuixStand (GE Healthcare, Little Chalfont, UK) and then filtered through a 0.22 μm bottle top filter (Sigma-Aldrich, St. Louis, MO). The filtrates were pelleted by ultracentrifugation in a 45 Ti rotor (Beckman Coulter, Fullerton, CA) at 100,000 × g for 2 h at 4°C. The final pellets were resuspended in phosphate-buffered saline (PBS) and stored at -80°C. We placed the EVs in PBS on 300-mesh copper grids and stained them with 2% uranyl acetate. Images were obtained using a JEM-1011 electron microscope (JEOL, Tokyo, Japan) operated at an acceleration voltage of 100 kV [27 (link), 28 (link)]. EV markers including CD81 and CD40 were analyzed by western blotting with primary antibodies, anti-CD81 (1 : 1000, Abcam, Cambridge, MA), and anti-CD40 (1 : 1000, Abcam) as previously described [22 (link)].
3
Comprehensive Immunophenotyping of Murine Cells
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The following Abs and reagents were used in this study: FITC anti-mouse CD3ε (#100306), FITC anti-mouse CD19 (#152404), APC anti-mouse CD21/CD35 (#123412), PerCP/Cyanine5.5 anti-mouse CD23 (#101618), and Brilliant Violet 421 anti-mouse IgD (#405725) (all from BioLegend, CA, USA); PE anti-mouse CD45 (#12-0451-82) (Invitrogen, CA, USA); anti-TCR α/β (#sc-19600), anti-CD4 (#sc-19641), and anti-GAPDH (#sc-365062) (Santa Cruz Biotechnology, TX, USA); anti-MHC Class II (#ab180779), anti-ICOS (#ab175401), anti-ICOSL (#ab138354), anti-CD40 (#ab188181), and anti-CD40L (#ab2391) (Abcam, Cambridge, UK); and FITC anti-mouse CD169 (MOMA-1, #MCA947F) (Bio-Rad, CA, USA).
4
Elucidating NF-κB Signaling in T-MSCs
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To determine the signaling pathways affected by priming with TNF-α and IFN-γ, T-MSCs were pretreated with the signaling pathway inhibitor, AMGEN16 (non-canonical NF-κB pathway, 5 μmol/L; Sigma-Aldrich). After pretreatment with an inhibitor, the cells were incubated with TNF-α and IFN-γ for 24 h and then lysed in RIPA buffer. The lysates were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with blocking solution and incubated with antibodies against phospho-p65 (#3033, Cell signaling Technology, Danvers, MA, USA), p65 (#8242, Cell signaling) of the canonical NF-κB pathway, and phospho-p100 (#4810, Cell signaling), p52 (#4882, Cell signaling), and RELB (#4922, Cell signaling) of the non-canonical NF-κB pathway (Cell signaling Technology, Danvers, MA, USA), an-ti-CD40 (# ab224639, abcam), and β-actin (#4970, Cell signaling Technology). The blots were visualized using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA).
5
Immunofluorescence Staining of Intestinal Tissues and Cells
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Intestinal tissues were collected and fixed overnight in 4% paraformaldehyde. The fixed intestinal tissues were OCT-embedded, snap-frozen, and prepared into 16 μm sections for immunofluorescence staining. Cell samples including the primary EGCs, primary astrocytes and Caco-2 cells were seeded in 24-well plates plated with cell climbing slices respectively. After stimulation, cell samples were fixed in 4% paraformaldehyde and washed thrice with PBS for immunofluorescence staining. After permeabilization and blocking, the tissue sections or cell samples were incubated with the respective primary antibodies overnight at 4 °C. Briefly, the primary antibodies consisted of chicken anti-GFAP (1:200, GeneTex, Irvine, CA, USA), rabbit anti-Iba-1 (1:200 Abcam, Cambridge, UK), rabbit anti-Sox10 (1:500, Abcam, Cambridge, UK), anti-CD40 (1:200, Abcam, Cambridge, UK), and ZO-1 (1:200, Abcam, Cambridge, UK) antibodies. Next, the tissue sections or cell samples were washed thrice with PBS and incubated with A488 anti-chicken (1:1000, GeneTex) or A594 anti-rabbit (1:1000, GeneTex) antibodies at 37 °C for 2 h. DAPI (1:1000, Invitrogen) was used for nuclei counterstaining. An Olympus FluoView FV1000 microscope (Olympus, Tokyo, Japan) was used to collect the fluorescent images of intestinal tissues and cell samples. Fluorescent intensity was analyzed by ImageJ.
6
CD40 Signaling in Macrophages
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CD40flflLysMcre and CD40flfl BMDMs were cultured as described above, treated for 10 min or 24 h with FGK45 (30 µg/ml), IFN‐γ (5 ng/ml) and LPS (100 ng/ml) and subsequently lysed in RIPA lysis buffer. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein samples were loaded on to a SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Bio‐Rad). After blocking with 5% BSA in PBS containing 0.05% Tween‐20, blots were incubated overnight with anti‐CD40 (Abcam, Cambridge, UK) and anti‐α‐tubulin (Cedarlane, Burlington, ON, Canada). Blots were then washed and incubated with the appropriate HRP‐conjugated secondary antibody (1:800; Thermo Scientific) and visualized using the SuperSignal West Pico Plus Chemiluminescent Substrate kit (Thermo Scientific).
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CD40flflLysMcre and CD40flfl BMDMs were cultured as described above, treated for 10 min or 24 h with FGK45 (30 µg/ml), IFN‐γ (5 ng/ml) and LPS (100 ng/ml) and subsequently lysed in RIPA lysis buffer. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein samples were loaded on to a SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Bio‐Rad). After blocking with 5% BSA in PBS containing 0.05% Tween‐20, blots were incubated overnight with anti‐CD40 (Abcam, Cambridge, UK) and anti‐α‐tubulin (Cedarlane, Burlington, ON, Canada). Blots were then washed and incubated with the appropriate HRP‐conjugated secondary antibody (1:800; Thermo Scientific) and visualized using the SuperSignal West Pico Plus Chemiluminescent Substrate kit (Thermo Scientific).
7
Immunofluorescence Staining of T-MSCs
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After the indicated treatments, T-MSCs were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min, permeabilized by incubation with a 0.05% Triton X-100 solution for 10 min, and blocked with 5% normal goat serum for 1 h. The cells were subsequently stained with specific primary antibodies against anti-CD40 (1:200; Abcam, Cambridge, MA, USA) and anti-RELB (1:100; Cell Signaling Technology, Danvers, MA, USA), followed by 2 h of incubation with Alexa 488 and Alexa 594-labeled secondary antibodies (1:1000; Molecular Probes, Eugene, OR, USA). For counterstaining, the nuclei were stained with DAPI. Images were captured using a confocal microscope (Carl Zeiss, Jena, Germany).
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