primary antibodies targeting anti-cd68  (Servicebio Inc)

Journal: Cell death & disease

Article Title: Chemotherapy-induced macrophage CXCL7 expression drives tumor chemoresistance via the STAT1/PHGDH-serine metabolism axis and SAM paracrine feedback to M2 polarization.

doi: 10.1038/s41419-025-07712-y

Figure Lengend Snippet: Fig. 1 CXCL7 upregulation in chemotherapy-resistant colorectal cancer. A Differential gene expression profiling between chemosensitive and chemoresistant colorectal tumors (TCGA cohort) treated with 5-FU/oxaliplatin combination therapy. B Representative immunohisto- chemical (IHC) staining of CXCL7 in chemotherapy-resistant versus chemotherapy-sensitive CRC specimens (Scale bar, 100 μm). C Gene Set Enrichment Analysis (GSEA) demonstrating cisplatin resistance pathway activation in CXCL7-high versus CXCL7-low expression subgroups. D Comparative histopathological analysis using H&E staining and IHC evaluation of CXCL7/CD68 expression in paired pre- and post- chemotherapy CRC specimens from individual patients (Scale bar, 100 μm). E CXCL7 protein localization by IHC in chemotherapy-treated CRC lesions versus matched adjacent normal mucosa (Scale bar, 100 μm). F Quantitative comparison of CXCL7 expression levels between chemotherapeutically treated malignant tissues and non-neoplastic colorectal tissues. G Stage-dependent IHC expression patterns of CXCL7 across colorectal cancer progression (Scale bar, 100 μm). All data are presented as mean ± SD. *P < 0.05; n.s.not significant.

Article Snippet: Primary antibodies targeting CD68 (1:500, Proteintech, 28058-1-AP), CXCL7 (1:500, Affinity, #DF6695), and CK (1:500, Affinity, #DF3072) were applied and incubated at room temperature for one hour.

Techniques: Gene Expression, Immunohistochemistry, Activation Assay, Expressing, Staining, Comparison

Journal: Cell death & disease

Article Title: Chemotherapy-induced macrophage CXCL7 expression drives tumor chemoresistance via the STAT1/PHGDH-serine metabolism axis and SAM paracrine feedback to M2 polarization.

doi: 10.1038/s41419-025-07712-y

Figure Lengend Snippet: Fig. 2 Macrophage-derived CXCL7 promotes chemoresistance in colorectal cancer cells. A CXCL7-associated single-cell analysis of CRC_GSE146771 using the TISCH2 database. B Correlation analysis of CXCL7 with macrophage infiltration via TCGA database. C Left: mIHC staining of chemotherapy-sensitive versus resistant CRC tissues showing CK+ (purple), CXCL7+ (white), and CD68+ macrophages (green), with DAPI nuclear staining (blue) (scale bar, 50 μm). Right: CXCL7+ macrophage proportions in chemotherapy-sensitive versus resistant patients. D Schematic of tumor cell-macrophage co-culture system. E CXCL7 mRNA and F CXCL7 protein levels in macrophages co-cultured with HCT116 cells ± 5-FU (5 μM), oxaliplatin (5 μM), or combination. F The expression of CXCL7 in chemotherapy-treated colorectal cancer tissues compared with non-tumor tissues was analyzed. G Representative images and quantification for colony formation of CRC cells with CXCL7 + Mø versus CXCL7- Mø under 5-FU/oxaliplatin treatment. H Annexin V/PI apoptosis rates and (I) TUNEL+ cells (green) in CRC cells co-cultured with CXCL7 + Mø versus CXCL7- Mø under chemotherapy (scale bar, 50 μm). All data are presented as mean ± SD. *P < 0.05; n.s.not significant.

Article Snippet: Primary antibodies targeting CD68 (1:500, Proteintech, 28058-1-AP), CXCL7 (1:500, Affinity, #DF6695), and CK (1:500, Affinity, #DF3072) were applied and incubated at room temperature for one hour.

Techniques: Derivative Assay, Single-cell Analysis, Staining, Co-Culture Assay, Cell Culture, Expressing, TUNEL Assay

Journal: Kidney international

Article Title: Kidney injury-mediated disruption of intestinal lymphatics involves dicarbonyl-modified lipoproteins.

doi: 10.1016/j.kint.2021.05.028

Figure Lengend Snippet: Figure 6 | Proteinuric kidney injury stimulates ileal macrophage production of vascular endothelial growth factor (VEGF)-C. (a) Puromycin aminoglycoside (PAN) increased ileal VEGF-C versus controls (Cont). (b) VEGF-C concentration in PAN lymph was lower, but total output of VEGF-C was significantly greater, in PAN versus Cont. (c) Double staining of ileum with VEGF-C (red) and cluster of differentiation (CD) 68 (green) showed a greater number of CD68-positive cells and CD68- and VEGF-C–positive cells (arrows) in PAN versus Cont. (d) Cultured macrophages exposed to isolevuglandin (IsoLG)–apolipoprotein AI (apoAI) expressed more VEGFC mRNA versus unmodified apoAI. In vivo, results are mean SD for 7 to 12 rats per group. In vitro, experiments were performed independently 3 times with 3 wells per treatment. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Article Snippet: Double staining for CD68 and VEGF-C used citrate buffer for antigen retrieval, followed by biotinylated primary antibody targeting CD68 (1:10; BioRad).

Techniques: Concentration Assay, Double Staining, Cell Culture, In Vivo, In Vitro

Journal: American Journal of Translational Research

Article Title: Activation of the CXCL16/CXCR6 pathway promotes lipid deposition in fatty livers of apolipoprotein E knockout mice and HepG2 cells

doi:

Figure Lengend Snippet: Establishment of inflamed NAFLD model. ApoE KO mice were fed with a normal diet containing 4% fat (Control), a high-fat diet containing 21% fat and 0.15% cholesterol (HF group), or a HF diet with 10% casein injection (HF+casein group) for 8 weeks (n=8). The levels of SAA in the serum of three groups were measured by enzyme linked immunosorbent assay (A). The results are expressed as the means ± SD (n=8). **P<0.01 vs. Control. The protein expression of CD68, TNF-α, and MCP-1 in the livers of the mice was measured by immunohistochemical staining (B, brown colour, original magnification ×400). The protein expression of TNF-α and MCP-1 in the livers of the mice was further checked by Western blotting. The identical total protein extracted from liver tissues was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-mouse polyclonal antibodies against TNF-α, MCP-1, or β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans of the protein bands from the mice in each group, normalised by comparison with β-actin (C and D). *P<0.05 vs. Control, **P<0.01 vs. Control. HepG2 cells were treated without (Control) or with 30 µg/ml of cholesterol (CHO group), 5 ng/ml of IL-1β (IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β (CHO+IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA (CHO+IL-1β + siCXCL16 group), or 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA negative control (CHO+IL-1β + sicontrol group) for 24 hours. Total RNA was extracted from the HepG2 cells and cDNA was aquired by reverse transcription. The mRNA expression of TNF-α and MCP-1 in HepG2 cells was determined by real-time PCR. β-actin served as the housekeeping gene (E). Results represent the means ± SD.**P<0.01 vs. Control, ##P<0.01 vs. CHO+IL-1β. The protein expression of TNF-α and MCP-1 in HepG2 cells was checked by Western blotting. The identical total protein extracted from the HepG2 cells was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-human polyclonal antibodies against MCP-1, TNF-α, or anti-human monoclonal antibody against β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans for TNF-α and MCP-1, normalised by comparison with β-actin (F and G). *P<0.05 vs. Control, **P<0.01 vs. Control, ##P<0.01 vs. CHO+IL-1β.

Article Snippet: Sections were then treated sequentially with normal nonimmune animal serum for 30 minutes and incubated with anti-mouse polyclonal primary antibodies targeting CD68 (Novus Biologicals Inc., Canada), monocyte chemotactic protein 1 (MCP-1, Santa Cruz Biotechnology Inc., USA), tumour necrosis factor-α (TNF-α, Santa Cruz Biotechnology Inc., USA), CXCL16 (R&D Systems Inc., USA), CXCR6 (Novus Biologicals Inc., Canada), ADAM10 (Abcam, UK), collagen I (Abcam, UK) and α-smooth muscle actin (α-SMA, Abcam, UK) at 4°C overnight.

Techniques: Control, Injection, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemical staining, Staining, Western Blot, Isolation, Nucleic Acid Electrophoresis, Comparison, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: American Journal of Translational Research

Article Title: Activation of the CXCL16/CXCR6 pathway promotes lipid deposition in fatty livers of apolipoprotein E knockout mice and HepG2 cells

doi:

Figure Lengend Snippet: Inflammation induced the activation of the CXCL16/CXCR6 pathway in hepatic cells. ApoE KO mice were fed with a normal diet containing 4% fat (Control) a high fat diet containing 21% fat and 0.15% cholesterol (HF group), or a high fat diet with a 10% casein injection (HF+casein group) for 8 weeks (n=8). The protein expression of CXCL16/CXCR6 pathway components in the three groups of mice was checked by immunohistochemistry (A I-IX, ×400) and Western blotting. The identical total protein extracted from liver tissues was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-mouse polyclonal antibodies against CXCL16, CXCR6, ADAM10, or β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans for the protein bands of CXCL16/CXCR6 pathway components, normalised by comparison with β-actin (B and C). **P<0.01 vs. Control. HepG2 cells were treated without (Control) or with 30 µg/ml of cholesterol (CHO group), 5 ng/ml of IL-1β (IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β (CHO+IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA (CHO+IL-1β + siCXCL16 group), or 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA negative control (CHO+IL-1β + sicontrol group) for 24 hours. Total RNA was extracted from the HepG2 cells and cDNA was acquired by reverse transcription. The mRNA expression of CXCL16, CXCR6, and ADAM10 in HepG2 cells was determined by real-time PCR. β-actin served as the housekeeping gene (D). Results represent the means ± SD. *P<0.05 vs. Control, **P<0.01 vs. Control, #P<0.05 vs. CHO+IL-1β, ##P<0.01 vs. CHO+IL-1β. The protein expression of CXCL16, CXCR6, and ADAM10 in HepG2 cells was checked by Western blotting. The identical total protein extracted from the HepG2 cells was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-human polyclonal antibodies against CXCL16, CXCR6, ADAM10, or anti-human monoclonal antibody against β-actin which was used as an internal control. The histogram represents means ± SD of the densitometric scans for CXCL16, CXCR6 and ADAM10, normalised by comparison with β-actin (E and F). *P<0.05 vs. Control, **P<0.01 vs. Control, #P<0.05 vs. CHO+IL-1β, ##P<0.01 vs. CHO+IL-1β. Immunofluorescent staining of CXCL16 and ADAM10 in HepG2 cells (G). The cells were stained with DAPI to visualise nuclei (blue), and with Alexa Fluor 488 and Alexa Fluor 594 to visualise the distribution of ADAM10 (green) and CXCL16 (red) proteins.

Article Snippet: Sections were then treated sequentially with normal nonimmune animal serum for 30 minutes and incubated with anti-mouse polyclonal primary antibodies targeting CD68 (Novus Biologicals Inc., Canada), monocyte chemotactic protein 1 (MCP-1, Santa Cruz Biotechnology Inc., USA), tumour necrosis factor-α (TNF-α, Santa Cruz Biotechnology Inc., USA), CXCL16 (R&D Systems Inc., USA), CXCR6 (Novus Biologicals Inc., Canada), ADAM10 (Abcam, UK), collagen I (Abcam, UK) and α-smooth muscle actin (α-SMA, Abcam, UK) at 4°C overnight.

Techniques: Activation Assay, Control, Injection, Expressing, Immunohistochemistry, Western Blot, Isolation, Nucleic Acid Electrophoresis, Comparison, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining

Journal: American Journal of Translational Research

Article Title: Activation of the CXCL16/CXCR6 pathway promotes lipid deposition in fatty livers of apolipoprotein E knockout mice and HepG2 cells

doi:

Figure Lengend Snippet: Effects of CXCL16/CXCR6 pathway activation on the expression of ECM components in the livers of ApoE KO mice and in HepG2 cells. ApoE KO mice were fed with a normal diet containing 4% fat (Control), a high fat diet containing 21% fat and 0.15% cholesterol (HF group), or with 10% casein injection (HF+casein group) for 8 weeks (n=8). Immunohistochemical staining shows the expression of collagen I and α-SMA in the livers of ApoE mice (A I-VI, ×400). The protein expression of collagen I and α-SMA in livers was checked by Western blotting. The identical total protein extracted from liver tissues was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-mouse polyclonal antibodies against collagen I, α-SMA, or β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans for the protein bands of collagen I and α-SMA, normalised by comparison with β-actin (B and C). **P<0.01 vs. Control. HepG2 cells were treated without (Control) or with 30 µg/ml of cholesterol (CHO group), 5 ng/ml of IL-1β (IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β (CHO+IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA (CHO+IL-1β + siCXCL16 group), or 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA negative control (CHO+IL-1β + sicontrol group) for 24 hours. Total RNA was extracted from the HepG2 cells and cDNA was acquired by reverse transcription. The mRNA expression of collagen I and α-SMA was determined by real-time PCR. β-actin served as the housekeeping gene (D). Results represent the means ± SD. *P<0.05 vs. Control, **P<0.01 vs. Control, #P<0.05 vs. CHO+IL-1β, ##P<0.01 vs. CHO+IL-1β. The protein expression of collagen I and α-SMA in HepG2 cells was checked by Western blotting. The identical total protein extracted from the HepG2 cells was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-human polyclonal antibodies against collagen I, α-SMA, or anti-human monoclonal antibody against β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans for the protein bands of collagen I and α-SMA, normalised by comparison with β-actin (E and F). **P<0.01 vs. Control, #P<0.05 vs. CHO+IL-1β, ##P<0.01 vs. CHO+IL-1β. HepG2 cells were washed with Dulbecco’s phosphate buffered saline and incubated in the dark for 6 hours with 50 μmol/l 5-(and-6)-chloromethyl-2’7’-dichlorodihydrofluorescein diacetate (green fluorescence) (G). The fluorescence intensities of ROS production was quantified by Cell Quest software. Data represent the means ± SD (H). **P<0.01 vs. Control, ##P<0.01 vs. CHO+IL-1β.

Article Snippet: Sections were then treated sequentially with normal nonimmune animal serum for 30 minutes and incubated with anti-mouse polyclonal primary antibodies targeting CD68 (Novus Biologicals Inc., Canada), monocyte chemotactic protein 1 (MCP-1, Santa Cruz Biotechnology Inc., USA), tumour necrosis factor-α (TNF-α, Santa Cruz Biotechnology Inc., USA), CXCL16 (R&D Systems Inc., USA), CXCR6 (Novus Biologicals Inc., Canada), ADAM10 (Abcam, UK), collagen I (Abcam, UK) and α-smooth muscle actin (α-SMA, Abcam, UK) at 4°C overnight.

Techniques: Activation Assay, Expressing, Control, Injection, Immunohistochemical staining, Staining, Western Blot, Isolation, Nucleic Acid Electrophoresis, Comparison, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Saline, Incubation, Fluorescence, Software