For isolation of naive neutrophils, peripheral blood of healthy donor or mice was collected and freshly isolated upon receipt. Separation of cells was undertaken by centrifugation over a three‐layer discontinuous Percoll gradient.50 Primary TANs were isolated from fresh colon cancer or xenograft specimens.7 In brief, fresh tissues were cut into small (1–2 mm) pieces, digested at 37°C for 2 h and subsequently filtered through cell strainers with diameters of 500, 100 and 70 µm. Cells were labelled with CD66b (ab233811) and CD11b (ab133357, Abcam Inc.) antibodies and purified by Microbeads (Miltenyi Biotec, Somerville, MA). For validating polarization of neutrophils, western blot was performed using antibodies specific for CD66b (ab233811) and CD11b (ab133357, Abcam Inc., Cambridge, MA). Neutrophils were grown in DMEM/F12 containing 0.02% bovine serum albumin, 10 mg·ml–1 apo‐transferrin and 1 mg·ml–1 insulin (Sigma) for 45 min. For co‐culture assay, 5 × 105 cancer cells were placed in lower compartment, while PNs or TANs (5 × 105 cells) were seeded into upper compartment of six‐well plates with 1.0 µm pore size inserts (Greiner‐Bio‐One, Kremsmünster, Austria).
Ab133357
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab133357 is a primary antibody that recognizes the protein of interest. It is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab182422, by Abcam (6 mentions)
Ab10558, by Abcam (6 mentions)
Ab16669, by Abcam (5 mentions)
Cd11b, by Abcam (4 mentions)
Ab64693, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ab183685, by Abcam (3 mentions)
Ab111101, by Abcam (3 mentions)
Ab238132, by Abcam (3 mentions)
Ab28364, by Abcam (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
F4 80, by BioLegend (2 mentions)
Ab208670, by Abcam (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Nb300 605, by Novus Biologicals (2 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (2 mentions)
Mca2314ga, by Bio-Rad (2 mentions)
90 protocols using ab133357
1
Isolation and Characterization of Neutrophils
2
Histological and Immunofluorescence Analysis
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For histological analysis, the dissected tissues were fixed in a mixture of 2% PFA and 20% sucrose solution for 24 h at room temperature, and then embedded in Tissue-tek O.C.T. (Electron Microscopy Sciences). Blocks were frozen in a dry ice and ethanol bath. For immunofluorescence, 6 μm O.C.T. tissue cryosections were stained with antibodies against α-SMA (ab5694, Abcam, 1: 200), CD11B (ab133357, Abcam, 1: 4,000), Ly6G (ab238132, Abcam, 1: 1,000), CD3 (ab133357, Abcam, 1: 100), CD206 (24595, CST, 1: 400), F4/80 (#30325, CST, 1: 800), CD86 (#91882, CST, 1: 800), SPP1 (#88742, CST, 1: 800). Secondary antibodies conjugated to Alexa Fluor® 488 conjugated-anti-rabbit IgG (H + L) (#4412, CST, 1: 2,000) and Alexa Fluor® 594 conjugated-goat antirabbit IgG (#8889, CST, 1: 2,000) were used. Nuclear staining was done with DAPI (40, 6-diamidino-2phenylindole). Immunofluorescence images were taken with a TCS SP8 STED 3X ultra-high resolution confocal system (Leica, Ernst-Leitz, Wetzlar, Germany).
3
Comprehensive Immunohistochemical Profiling
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Immunohistochemistry (IHC) staining was performed according to a previous method [13 (link), 20 ]. Serial paraffin sections were subjected to antigen retrieval, incubation in antigen retrieval solution for 20 min, inactivation with endogenous peroxidase (3% H2O2), and blocked in goat serum for 1 h. Sections were then incubated with primary antibodies against CD3 (SC-20047, Santa Cruz Biotechnology Inc., Dallas, TX, USA), F4/80 (SC-377009, Santa Cruz Biotechnology Inc.), IL-6 (GeneTex, Santa Cruz Biotechnology Inc.), IL-1β (ab9722, Abcam), TNF-α (ab183218, Abcam), p-p65 (Ser536) (#3033, Cell Signaling Technology), p16 (ab211542, Abcam), NLRC4 (ab201792, Abcam), ITGAM (ab133357, Abcam), p19 (ab80, Abcam), Collagen I (14695-1-AP, Proteintech), Collagen III (22734-1-AP, Proteintech), α-SMA (14395-1-AP, Proteintech), and β-galactosidase (15518-1-AP, Proteintech). After washing, sections were incubated with a secondary antibody for 1 h, and processed using the SABC-POD kit (SA2001, Boster, China). Then, sections were counterstained with Hematoxylin and fixed with biomount medium. Hematoxylin and Shandon Instant Eosin (Solarbio Co., Ltd.) were used to determine cell infiltration. Masson's trichrome staining (Sigma-Aldrich®) was used to assess collagen deposition.
4
Immunofluorescence Staining of Paraffin-Embedded Tissues
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All tissues were fixed in 10% formalin, sectioned, and embedded in paraffin or underwent H&E staining (Mayo Clinic Histology Core Facility). Immunofluorescence staining was performed based upon established protocols [28 ]. Briefly, slides were deparaffinized in a series of washes of decreasing ethanol content. CD3, GFP, CD11b-stained slides underwent heat-mediated antigen retrieval using sodium citrate buffer, while VV-stained slides used Tris/EDTA buffer. Slides were then stained with anti-CD3 (ab16669, Abcam, Cambridge, MA), anti-VV (ab35219, Abcam, Cambridge, MA), anti-GFP (ab6556, Abcam, Cambridge, MA), or anti-CD11b (ab133357, Abcam, Cambridge, MA) antibodies, followed by secondary staining with an AF568-tagged goat anti-rabbit antibody (A11011, Invitrogen, Carlsbad, CA) and counterstaining with DAPI. Images were acquired with an LSM780 confocal microscope and Zen software (Carl Zeiss, Thornwood, NY). Quantification was performed using ImageJ for tumor area calculation and blinded manual counting of CD3+ cells.
5
Biodistribution of Nanoparticles in Tumor-Bearing Rats
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The subcutaneous Walker-256-bearing rats were administered FITC-tagged bE-NPs or FITC-tagged s-NPs via tail vein injection. In the FITC-tagged bE-NPs group, the blood was taken from rats and the FITC-tagged bE-NPs were prepared as described above. After 12 h postinjection, these rats were anesthetized and perfused with saline and 4% paraformaldehyde. The tumors were harvested and fixed in 4% paraformaldehyde and then dehydrated with sucrose solution for 24 h and embedded in OCT (Sakura, Torrance, CA, USA). The tumors were cut into 5 mm by frozen sectioning and subjected to fluorescence microscope after staining with DAPI (blue) for the distribution of FITC-tagged bE-NPs (green) in tumor. For analyzing the tumor-infiltrated macrophages, sections were incubated with Ribbit monoclonal antibodies recognising CD11b (ab133357, Abcam) and Mouse monoclonal antibodies recognising CD68 (ab201340, Abcam), followed by staining with AlexaFluor 568-conjugated Goat Anti-Ribbit IgG secondary antibody (shown in red) and AlexaFluor 647-conjugated Goat Anti-Mouse IgG secondary antibody (shown in violet). The images were captured by fluorescence microscope and presented as visions on the site with a tumor penetration depth of 2 mm.
6
Immunohistochemical Visualization of Immune Markers
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The avidin–biotin immunoperoxidase method was used for deparaffinized zinc formalin-fixed, paraffin-embedded sections. Specific methods are detailed in our previous article (27 (link)). The primary antibodies including CD11b (1:5,000, ab133357, Abcam), F4/80 (1:200, 123101, BioLegend), CD68 (1:500, GB11067, Servicebio), Cleaved caspase-3 (1:300, 9661, Cell Signaling), BrdU (1:50, B44, BD), Ki67 (1:400, 12202, Cell Signaling), CD8 (1:500, GB11068, Servicebio), granzyme-B (1:200, sc-8002, Santa Cruz), followed by incubation with secondary antibodies: biotinylated goat anti-mouse IgG (1:300, BA-9200, Vector), biotinylated goat anti-rat IgG (1:300, BA-9400, Vector) and biotinylated goat anti-rabbit IgG (1:300, BA-1000, Vector). Images were taken through a light microscope (Olympus).
7
Immunohistochemical Analysis of Colon Tissue
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Colon tissue sections (4 mm) were deparaffinized in xylene twice for 10 min, rehydrated in a graded ethanol series once for 5 min, incubated in sodium citrate buffer for 10 min for antigen retrieval, blocked with 10% bovine serum albumin for 1 h, and incubated with antibodies against Claudin-1 (1:100; Abcam, Cambridge, UK; ab242370), zonula occludens 1 (ZO-1) (1:100; Abcam; ab276131), Occludin (1:100; Abcam; ab216327), mucin 2 (MUC2) (1:500; Abcam; ab272692), myeloperoxidase (MPO) (1:500; Abcam; ab208670), CD11b (1:100; Abcam; ab133357), IL-17 (2 mg/mL; Abcam; ab79056), IL-10 (10 mg/mL; Abcam; ab189392) overnight, and IL-1β (1:500; Abcam: ab283818). The sections were then incubated with secondary antibodies (SP9000; Zsbio Biotechnology, Beijing, China) for 1 h. The images were acquired using a BX53F microscope (Olympus, Tokyo, Japan).
8
Multimodal Assessment of Brain Pathology
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Cryosections of the brains were stained with DAPI (4′,6-diamidino-2-phenylindole) and evaluated by fluorescence microscopy. Disturbance of the BBB was visually evaluated using Evans blue dye (EBD). Therefore, 500 μl/kg 2% EBD was injected i.v. 30 min before decapitation of the rat for AR. EBD extravasation in brain slices was examined by fluorescence microscopy (LMD6000, Leica Microsystems CMS GmbH) and processed by AIDA software (AIDA Version 4.50, Raytest).
Blood vessels were stained with anti-rat von Willebrand factor antibody (ab6994, Abcam). To visualize activated microglia, anti-rat CD11b (Integrin alpha M) antibody (ab133357, Abcam) was used. Reactive astrocytes were visualized by staining with anti-rat GFAP (glial fibrillary acidic protein) antibody (ab53554, Abcam). Immunofluorescence PSMA staining was applied to tumor bearing brain slices using three different anti-PSMA antibodies (NBP1-45057 and NBP1-89822, Novus Biologicals; ab58779, Abcam) to display PSMA expression. To verify the functionality and specificity of the used PSMA antibodies, rat prostate and kidney tissue were used as positive controls. Immunofluorescence stainings were performed according to standard histology protocols and as described before [19 (link)].
Blood vessels were stained with anti-rat von Willebrand factor antibody (ab6994, Abcam). To visualize activated microglia, anti-rat CD11b (Integrin alpha M) antibody (ab133357, Abcam) was used. Reactive astrocytes were visualized by staining with anti-rat GFAP (glial fibrillary acidic protein) antibody (ab53554, Abcam). Immunofluorescence PSMA staining was applied to tumor bearing brain slices using three different anti-PSMA antibodies (NBP1-45057 and NBP1-89822, Novus Biologicals; ab58779, Abcam) to display PSMA expression. To verify the functionality and specificity of the used PSMA antibodies, rat prostate and kidney tissue were used as positive controls. Immunofluorescence stainings were performed according to standard histology protocols and as described before [19 (link)].
9
Multiplex Immunostaining of Tumor Microenvironment
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H&E staining was performed on the TMA specimens, and the results were assessed by an experienced pathologist. For the immunohistochemistry (IHC) staining, anti-CD8 (Cell Signaling Technology, Danvers, Massachusetts, USA) and anti-FoxP3 (R&D Systems, Tustin, California, USA) monoclonal antibodies were utilized, as previously described.20 21 (link) The average number of tumor-infiltrating immune cells per high-power fields (HPF) were assessed by three independent ×400 HPF.
Immunofluorescence (IF) staining was performed on the TMA specimens of FFPE tissue. In this study, we used anti-myeloperoxidase (MPO) (Abcam, ab208670, Cambridge, UK), anti-CD11b (Abcam, ab133357), and anti-CD206 (Abcam, ab64693) monoclonal antibodies as the primary antibodies at room temperature for 1 hour of incubation. Next, the specimens were incubated for an additional hour with Alexa Fluor 488 (B40953), Alexa Fluor 555 (B40955), and Alexa Fluor 647 (B40958) along with Tyramide Reagent (ThermoFisher Scientific, Waltham, Delaware, USA). As the final step, the specimens were incubated for 20 min with DAPI (Solarbio, #C0060). A fluorescence microscope (Olympus, Tokyo, Japan) was used to visualize the cells.
Immunofluorescence (IF) staining was performed on the TMA specimens of FFPE tissue. In this study, we used anti-myeloperoxidase (MPO) (Abcam, ab208670, Cambridge, UK), anti-CD11b (Abcam, ab133357), and anti-CD206 (Abcam, ab64693) monoclonal antibodies as the primary antibodies at room temperature for 1 hour of incubation. Next, the specimens were incubated for an additional hour with Alexa Fluor 488 (B40953), Alexa Fluor 555 (B40955), and Alexa Fluor 647 (B40958) along with Tyramide Reagent (ThermoFisher Scientific, Waltham, Delaware, USA). As the final step, the specimens were incubated for 20 min with DAPI (Solarbio, #C0060). A fluorescence microscope (Olympus, Tokyo, Japan) was used to visualize the cells.
10
Immunohistochemical Analysis of Tumor Markers
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Formalin-fixed, paraffin-embedded, tumor sections (4 μm) were cut with a microtome, mounted on ChemMateTM capillary gap slides (Dako, Glostrup, Denmark), dried at 60 °C, deparaffinized, and hydrated. Hydrogen peroxide (1.5%) in TBS buffer (pH 7.4) was used for blocking endogenous peroxidase, and antigen retrieval was achieved by microwave boiling in TEG buffer (Dako) for 15 min. Sections were incubated with the primary antibody: anti-mouse CD11b (ab133357, Abcam); anti-human cytokeratin (M3515, Dako, Clone AE1/AE3); and anti-human CD8 (108M, Sigma-Aldrich) at room temperature for 32 min before detection with the OptiView DAB IHC detection kit (760-700, Vienna Medical Systems).
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