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12 protocols using f4 80

1

Quantifying Colon Inflammation via F4/80 Immunohistochemistry

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F4/80 positive inflammatory cell infiltration analysis was performed on paraffin-embedded colon tissue sections. The sections were deparaffinized, rehydrated with xylene and graded ethanol solutions, and washed with PBS. After blocking with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at 37°C, the sections were incubated with primary antibodies against F4/80 (cat. no. 14-4801-81; eBioscience; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at a dilution of 1:50 for 2 h at 37°C, washed with PBS and subsequently co-incubated with biotinylated secondary antibody (cat. no. KS010; Nanjing Jiancheng Bioengineering Institute) for 30 min at room temperature at a dilution of 1:2,000. Following washing with PBS (pH 7.4), tissue sections were incubated at 37°C with a horseradish peroxidase (HRP)-streptavidin complex (cat. no. KS001; Nanjing Jiancheng Bioengineering Institute) to detect secondary antibody for 30 min. Sections were stained with DAB at room temperature for 5 min and mounted according to standard protocols and observed under a light microscope at magnification, ×20.
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2

Histological Analysis of Post-MI Inflammation

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At 3 days post-MI, hearts were harvested, post-fixed with 4% paraformaldehyde for 12 h at 4°C and sliced horizontally. According to the manufacture's protocol, infiltrated neutrophils in heart sections (5 µm) were stained with naphtol AS-D chloroacetate esterase kit (cat. no. 90C2; Sigma-Aldrich; Merck KGaA) followed by hematoxylin (concentrations not commercially available) at 25°C for 10 sec. Accumulation of macrophages in the ischemic hearts was evaluated using the macrophage specific antibody F4/80 (1:50; cat. no. 70076; Cell Signaling Technology, Inc.), followed by counterstaining with 100 µl hematoxylin (concentration not commercially available) (cat. no. C0107; Beyotime Biotechnology, Inc) at 25°C for 10 sec. A total of four independent areas of each section were examined using light microscopy at a magnification of ×400.
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3

Cryosectioning and Immunostaining of Enucleated Eyeballs

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Each enucleated eye ball was fixed with paraformaldehyde and then placed in a slurry of optimal cutting temperature (OCT) compound in cryomold before freezing in dry ice and storage in a − 80 °C freezer until ready for sectioning using the Microm HM550 (Carl Zeiss Ltd). Five-micrometer cryosections of day 2 post-operated eye tissues stained with hematoxylin and eosin (H&E) staining was visualized as described previously [14 (link)]. A total of 3 eyes for each condition were evaluated. Acetyl-histone H3, CD45 and F4/80 antibodies were obtained from Merck Millipore (Darmstadt, Germany), BD Pharmingen (San Diego, CA, USA) and Abcam Plc (Cambridge, UK), respectively. Isolectin B4-Alexa Fluor 568 conjugate was from Molecular Probes Inc. (Eugene, OR, USA). Labeling by the primary antibodies was detected using secondary antibodies conjugated to Alexa Fluor-594 (red fluorescence) or Alexa Fluor-488 (green fluorescence), both obtained from Invitrogen Corp. (Thermo Fisher Scientific Inc., Waltham, MA, USA). Nuclei were visualized by mounting the cells in DAPI-containing Vectashield mounting medium (Vector Laboratories, CA, USA). Labeled cells were visualized using the Zeiss Imager.Z1 microscope (Carl Zeiss Inc., USA).
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4

Molecular Mechanisms of Anti-Inflammatory Actions

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F4/80 (#SAB5500103) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The TLR4 inhibitor TAK-242 (#614316) was obtained from Calbiochem (San Diego, CA, USA). The PI3K inhibitor wortmannin (#HY-10197) was obtained from MedChemExpress (New Jersey, USA). Collagen II (#28459-1-AP) was obtained from Proteintech Group (Wuhan, China). TLR4 (#AF7017), CD68 (#DF7518), PI3K (#DF6069) antibodies were obtained from Affinity Biosciences (Cincinnati, OH, USA). SP-D (#ab220422), MD-2 (#ab24182), TNF-α (#ab66579), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#ab181602), MMP-13 (#ab84594), and NLRP3 (ab263899) antibodies were obtained from Abcam (Cambridge, UK). Antibodies for NF-κB p65 (#8242), phospho-p65 (p-p65) (#3033), p-Akt (#4060) and Akt (#9272) were procured from Cell Signaling Technology (Boston, MA, USA). Recombinant human SP-D (rhSP-D) (#CSB-YP021175HU) was purchased from Huamei Biotech Co., Ltd (Wuhan, China). ELISA Kits of rat IL-1β (#ELK1272) and TNF-α (#ELK1396) were purchased from ELK Biotechnology (Wuhan, China). SNP (#1008) was purchased from Youcare Pharmaceutical Group Co., Ltd (Beijing, China). All of the other chemicals and reagents were of analytical grade.
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5

Murine Immune Cell Phenotyping

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Spleens were mashed between two glass slides and passed repeatedly through a 25 5/8G needle to dissociate single cells. Following RBC lysis, cells were quantified using a Countess(Invitrogen). Lymph nodes were processed as above without RBC lysis. Cells stained using these antibodies(Biolegend): B220(RA3-6B2), CD19(6D5), Kappa(RMK-45), Lambda(RML-42), CD11c(N418), CD8α(53-6.7), CD3ε(145-2C11), CD4(GK1.5), CD62L(MEL-14), CD44(IM7), CD25(PC61), Pan-NK(DX5), CD11b(M1/70), Ly6G(1A8), CD115(AFS98), F4/80(BM8), and DAPI(Sigma).
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6

Quantifying Wound Angiogenesis and Inflammation

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To measure the CD31 and F4/80 protein levels, mice were sacrificed and wound tissues were harvested 7 days after treatment. Briefly, the harvested wound tissues were homogenized using Multi-beads shocker® and added to the T-PER Reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Total protein lysates were obtained after the elimination of debris by filtering with 0.22 μm filters (Millipore). Total protein (10 μg) was separated using a mini-protean TGX Stain-FreeTM gel (BioRad), and then transferred to PVDF membranes using the Trans-Blot® TurboTM transfer system (BioRad). After blocking with the PVDF Blocking Reagent Can Get Signal® (TOYOBO) for 1 hour at room temperature, the membranes were incubated with primary antibodies against CD31, F4/80, or tubulin (Sigma-Aldrich) at 4 °C overnight, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Expression was visualized by chemiluminescence with a digital luminescent image analyzer (LAS-4000, GE Healthcare).
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7

Immunofluorescence Analysis of Pancreatic Tissues

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The mouse pancreatic tissues were harvested 1, 2, or 4 weeks after MSC infusion. First, the mice anaesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) were perfused with PBS through the left ventricle, followed by 4% paraformaldehyde. Then, the pancreases were isolated, dehydrated with 30% sucrose/PB overnight, and embedded in optimal cutting temperature compound (OCT). Pancreatic sections (6 mm) were sliced by a microtome (Thermo Fisher Scientific) and incubated in a humidified chamber at 4°C overnight with primary antibodies against insulin (1/200, guinea pig, Sigma-Aldrich), glucagon (1/2,000, mouse, Abcam), Pdx1 (1/200, rabbit, CST), CD11c (1/200, mouse, Abcam), IL1β (1/100, rabbit, Abcam), F4/80 (1/200, rabbit, Sigma-Aldrich), and Fizz1 (1/200, rabbit, Abcam). After the sections were washed with PBS, they were incubated for 2 h with a secondary antibody (1 : 500; Alexa Fluor 488/594-conjugated secondary antibodies, Invitrogen) at room temperature. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich). The images were captured with a confocal laser scanning microscope (Olympus, Tokyo, Japan). Peritoneal macrophages spread on glass coverslips were fixed with 4% paraformaldehyde. The remaining steps were performed as described above.
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8

Immunocytochemistry and Immunohistochemistry of Spinal Cord Injury

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For immunocytochemistry, OECs and microglia were washed with PBS, and fixed in 4% PFA for 20 min at room temperature. After blocked with 3% BSA for 15 min, cells were stained with primary antibodies: P75NTR (ab52987, 1:500; Abcam), iNOS (ab49999, 1:200; Abcam), F4/80 (SAB5500103, 1:100; Sigma), p‐c‐Jun (3270s, 1:5000; Cell Signaling Technology), and p‐NF‐κB (3033t, 1:2000; Cell Signaling Technology) at 4°C overnight. The cells were then stained with corresponding secondary antibodies. A confocal microscope (LSM 800; Zeiss) was utilized for examination and photography.
For immunohistochemistry, slides of injured spinal segments were blocked in 0.01 M PBS containing 3% BSA and 0.1% Triton X‐100 for 20 min. Subsequently, slides were incubated with primary antibodies: Iba1 (019‐19741, 1:1000; Wako), iNOS (ab49999, 1:200; Abcam), Arginase1 (sc‐166920, 1:200; Santa Cruz Biotechnology), GFAP (1:2000; Sigma), NeuN (#24307, 1:200; Cell Signaling Technology) at RT overnight. The slides were then stained with corresponding secondary antibodies, and counterstained with Hoechst 33342 to display nuclei. Slides were photographed by a confocal microscope (LSM 800; Zeiss).
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9

Quantification of Iron Homeostasis Markers

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Assays were performed with power SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA) as previously described50 (link) using the ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Primers for quantification of HIF-2α, CRP, Hepcidin, IL-6, EPOR, TFR, c-MYC, IL-1β, FPN-1, IRAK-4, MYD-88, TGF-β, CD-163, CD-11c, F4/80, Ferritin, TNF-α, IL-10 and β-actin (Sigma-Aldrich, Rehovot, Israel) are summarized in Supplemental Table 2.
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10

Fluorescent Immunohistochemistry for Cell Identification

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Fluorescent immunohistochemistry was performed on cryostat sections using the method as described previously [23 (link)]. Primary mouse monoclonal, rat monoclonal or rabbit polyclonal antibodies raised against calbindin (Swant, Marly, Switzerland; 1:1000), CD68 (Serotec, Raleigh, NC; 1:500), glial fibrillary acidic protein (GFAP; DAKO, Carpinteria, CA; 1:300), glutamine synthetase (Abcam, Cambridge, MA; 1:1000), alpha subunit of G protein (Goα; Millipore, Billerica, MA; 1:1000), hexaribonucleotide binding protein-3 (NeuN; Millipore; 1:100), Neurofilament (SMI32; Covance, Munich, Germany; 1:1000), Npc1 (Abcam; 1:300), alpha isoform of protein kinase C (PKCα; Sigma; 1:1000), tyrosine hydroxylase (Millipore; 1:1000), and a microglia marker (F4/80; 1:200) were used. All primary antibodies are specific to determine their target proteins, which were used as specific markers to identify different types or structures of cells. Alexa 488-labeled (Molecular Probes, Eugene, USA) or Cy3-labeled (Dianova, Hamburg, Germany) secondary antibodies against mouse, rat or rabbit IgG were used. Fluorescence in sections was detected by a confocal microscope (Zeiss LSM780; Jena, Germany). Digital images were adjusted in contrast and brightness with the Photoshop software (Adobe Systems, San Jose, USA).
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