Tissues were harvested, washed with cold PBS buffer and fixed for either paraffin embedding or OCT embedding. H&E, Masson’s trichrome and LY-6G immunohistochemistry (IHC, 1:200, #87048s, Cell Signaling Technology, US) staining were carried out. For frozen tissue sections, after permeabilization and blocking, tissue slides were probed with the following primary antibodies: CX3CR1 (1:200, ab8201, Abcam, US), CCR2 (1:200, ab203128, Abcam, US), MPO (1:200, ab208670, Abcam, US). After washes with PBS, samples were processed with fluorescent secondary antibodies for 1 h, followed by DAPI staining for 10 min to visualize nucleus. Images were visualized with fluorescence microscope (Carl Zeiss, Germany).
Ab203128
Manufactured by Abcam
Sourced in United States
Ab203128 is a lab equipment product from Abcam. It is a core component used for [CORE FUNCTION].
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (2 mentions)
Ab208670, by Abcam (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Ab8201, by Abcam (1 mentions)
Rnaeasy animal rna isolation kit, by Beyotime (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Ab76967, by Abcam (1 mentions)
Rpn800e, by GE Healthcare (1 mentions)
T9026, by Merck Group (1 mentions)
Ab179471, by Abcam (1 mentions)
Ab175388, by Abcam (1 mentions)
Ab81280, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Ab53444, by Abcam (1 mentions)
Ab222783, by Abcam (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab7778, by Abcam (1 mentions)
Ab6640, by Abcam (1 mentions)
10 protocols using ab203128
1
Multiparametric Tissue Analysis Protocol
2
Quantifying Inflammatory Markers in Murine Models
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Anti-mouse CCL2 (#2029S) was purchased from Cell Signaling Technology. Anti-mouse CCR2 (ab203128) and TCR-γ/δ (ab231545) were purchased from Abcam Co., LTD. The enzyme-linked immunosorbent assays (ELISA) kits were purchased from Invitrogen Co., LTD. RNAeasy™ Animal RNA Isolation Kit was purchased from Beyotime Co., LTD. ReverTra Ace qPCR RT Kit and SYBR® Green Realtime PCR assay kits were purchased from TOYOBO Co., LTD.
3
Intestinal Epithelial Protein Analysis
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Small intestinal crypts and villi were extracted as described above. The epithelium was then pelleted 10 min at 500 × g, resuspended in Laemmli 2 × lysis buffer supplemented with 5% beta-mercaptoethanol and boiled for 1 min. Samples were briefly sonicated to reduce viscosity.
Antibodies against alpha-Tubulin (T9026, Sigma), Strc (Biorbyt orb313204, 1 µg/ml), Ddo (Abcam ab175110, 0.8 µg/ml), Ccr2 (ab203128, 0.66 µg/ml), Ddr2 (Abcam ab76967, 1.8 µg/ml) were used for Western blot with 3% BSA blocking, tris- buffered saline-0.1% Tween-20 (TBS-T) washing buffer and enhanced chemiluminescence (ECL) detected on x-ray film. Protein ladders were from Thermo Scientific #26616 and from GE Healthcare #RPN800E. Protein band intensity was quantified and calculated relative to their corresponding loading control signals (alpha-tubulin) after background subtraction with FIJI-software47 (link) (Supplementary Table3 ).
Antibodies against alpha-Tubulin (T9026, Sigma), Strc (Biorbyt orb313204, 1 µg/ml), Ddo (Abcam ab175110, 0.8 µg/ml), Ccr2 (ab203128, 0.66 µg/ml), Ddr2 (Abcam ab76967, 1.8 µg/ml) were used for Western blot with 3% BSA blocking, tris- buffered saline-0.1% Tween-20 (TBS-T) washing buffer and enhanced chemiluminescence (ECL) detected on x-ray film. Protein ladders were from Thermo Scientific #26616 and from GE Healthcare #RPN800E. Protein band intensity was quantified and calculated relative to their corresponding loading control signals (alpha-tubulin) after background subtraction with FIJI-software47 (link) (Supplementary Table
4
Protein Characterization via SDS-PAGE and Western Blot
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The retained proteins were characterized by polyacrylamide gel electrophoresis (SDS-PAGE). The protein samples of MM@PCM/RAP, macrophage membrane (MM) and macrophage were extracted by RIPA lysis buffer contained PMSF. After adding the loading buffer, the protein samples were transferred to a metal bath at 95 °C for 10 min to fully denature the protein. Subsequently, 20 μL of each sample was loaded onto the pre-configured 10% gel. Gels were run at 140 V for 60 min to separate protein bands of different molecular weights. Bands were visualized using Coomassie brilliant blue.
The CCR2, integrin α4, integrin β1 and CD47 on the cell membrane were characterized by western blot (WB). Briefly, after separate protein bands in 8% gel, the proteins were transferred onto a PVDF membrane at 90 mA for 90 min. The PVDF membrane was blocked with 5% milk for 2 h and then incubated overnight at 4 °C with the primary antibody of CCR2 (1:1000, ab203128, Abcam), integrin α4 (1:1000, ab81280, Abcam), integrin β1 (1:1000, ab179471, Abcam) and CD47 (1:1000, ab175388, Abcam). The following day, membranes were incubated with an HRP-conjugated secondary antibody (1:5000, ab6721, Abcam) for 1 h and observed by ChemiDoc XRS imaging system (Bio-Rad, USA).
The CCR2, integrin α4, integrin β1 and CD47 on the cell membrane were characterized by western blot (WB). Briefly, after separate protein bands in 8% gel, the proteins were transferred onto a PVDF membrane at 90 mA for 90 min. The PVDF membrane was blocked with 5% milk for 2 h and then incubated overnight at 4 °C with the primary antibody of CCR2 (1:1000, ab203128, Abcam), integrin α4 (1:1000, ab81280, Abcam), integrin β1 (1:1000, ab179471, Abcam) and CD47 (1:1000, ab175388, Abcam). The following day, membranes were incubated with an HRP-conjugated secondary antibody (1:5000, ab6721, Abcam) for 1 h and observed by ChemiDoc XRS imaging system (Bio-Rad, USA).
5
Immunofluorescence Analysis of Cardiac Tissue
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Cardiac tissue sections were selected for further immunofluorescence analysis as previously described (22 (link)). The paraffin sections were deparaffinized, hydrated, and then subjected to sodium citrate antigen retrieval. The subsequent steps were similar to those used for processing the frozen sections. Specifically, after rinsing with PBST (phosphate-buffered saline with Tween 20), nonspecific binding was blocked with 5% bovine serum albumin. The sections were then incubated overnight at 4°C with specific primary antibodies. The following primary antibodies were used: α-SMA (A5228; Sigma-Aldrich, St. Louis, MO, USA), collagen 1a1 (NBP1-30054; Novus Biologicals, Littleton, CO, USA), collagen 3a1 (ab7778, Abcam), F4/80 (ab6640, Abcam), CD68 (ab53444, Abcam), VEGF (sc-7269, Santa Cruz Biotechnology, CA, USA), Ki67 (ab15580; Abcam), CCR2 (ab203128, Abcam), cardiac troponin I (TNNI3) (SAB2502170, Sigma-Aldrich), CD31 (ab222783, Abcam), and BDNF (ab108319, Abcam). Sections were incubated with fluorescent secondary antibodies for 2 hours at room temperature. After 4′,6-diamidino-2-phenylindole staining, the sections were treated with antiquenching reagent and observed under a laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). For each section, 10 to 15 fields were randomly selected, and the Zeiss software system was used for subsequent image processing.
6
Quantifying CCR2 Expression in Lumbar DRG Neurons
7
Membrane Protein Characterization by SDS-PAGE
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Membrane proteins were characterised by polyacrylamide gel electrophoresis (SDS-PAGE). Collected membrane proteins received using Bio-Rad electrophoresis system were run on 4- to 12% Bis–Tris 10-well mini-gels at 75 V for 0.5 h, followed by 140 V for 1 h. Finally, visualization of the resulting polyacrylamide gels was carried out by overnight staining with SimplyBlue.
The amount of CCR2 and CD47 in the samples was determined by Western blot analysis. Total MM protein extracted from 1 × 107 RAW 264.7 cells and subsequently MM@MTX NPs, was extracted using the Total Cell Protein Extraction Kit. Samples were electrophoresed on 10% SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, USA). Membranes were then treated with primary anti-CCR2 (Abcam, anti-CCR2 antibody ab203128, USA) and CD47 (Abcam, anti-CD47 antibody ab175388, USA) followed by horseradish peroxidase-labelled goat/anti-rabbit IgG (H + L) (Beyotime, Jiangsu, China). Protein signals were measured by enhanced chemiluminescence and visualized with a ChemiDoc MP imaging system (Bio-Rad, ChemiDoc MP, USA).
The amount of CCR2 and CD47 in the samples was determined by Western blot analysis. Total MM protein extracted from 1 × 107 RAW 264.7 cells and subsequently MM@MTX NPs, was extracted using the Total Cell Protein Extraction Kit. Samples were electrophoresed on 10% SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, USA). Membranes were then treated with primary anti-CCR2 (Abcam, anti-CCR2 antibody ab203128, USA) and CD47 (Abcam, anti-CD47 antibody ab175388, USA) followed by horseradish peroxidase-labelled goat/anti-rabbit IgG (H + L) (Beyotime, Jiangsu, China). Protein signals were measured by enhanced chemiluminescence and visualized with a ChemiDoc MP imaging system (Bio-Rad, ChemiDoc MP, USA).
8
Protein Isolation and Western Blot Analysis
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Proteins were isolated from cells using radioimmunoprecipitation assay buffer and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer onto polyvinylidene fluoride membranes, samples were blocked with Tris-buffered saline containing Tween 20 (TBST) and 5% bovine serum albumin. Thereafter, membranes were incubated with diluted primary antibodies, as follows: anti-β-actin (ab179467, 1:5,000; Abcam), anti-TGFα (ab92486, 1:500; Abcam), anti-CCL2 (#2029, 1:1,000; Cell Signaling Technology (CST), USA), or anti-CCR2 (ab203128, 1:1,000; Abcam), overnight at 4°C. Subsequently, horseradish peroxidase-conjugated secondary antibody (ab150077, 1:1,000; Abcam) was applied and the membrane was developed using chemiluminescence reagent. Protein levels were quantified using ImageJ software (National Institutes of Health (NIH), USA). Relative standard error (SE) was measured by dividing the densitometry value of the target protein by that of the respective loading control.
9
Western Blot Analysis of Macrophage Proteins
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Macrophages were lysed with radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz) for protein extraction. Then, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE, 10%) was performed to separate the proteins, which were then transferred to PVDF membranes (Millipore). The membranes were incubated overnight with primary antibodies against phospho-p65 (1:1000, #93H1; CST), p65 (1:1000, #8242; CST), CCR2 (1:1000, ab203128; Abcam), or β-actin (1:2000, #4970; CST). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Shengxing Biological) before visualization using enhanced chemiluminescence (Millipore).
10
Fluorescence Immunostaining of Immune Cells
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For fluorescence immunostaining, cells grown on coverslips or frozen tissue sections were routinely fixed, permeabilized and blocked as we described previously. 7 Fixed slides were further stained with anti-F4/80 (Abcam, ab6640, 1 : 100), anti-CD11b (Biolegend, 101201, 1 : 100), or anti-CCR2 (Abcam, ab203128, 1 : 100), and detected by fluorescent conjugated secondary antibodies (Yeasen). Nuclei were counterstained with DAPI (Solarbio). Images were captured using a confocal microscope (Zeiss LSM880) and processed using the ZEN software.
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