Journal: Biology of reproduction

Article Title: Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content.

doi: 10.1095/biolreprod61.3.705

Figure Lengend Snippet: FIG. 1. Light micrographs showing sec- tions of the epididymis of the Brown Nor- way rat stained with an antibody for monocytes-macrophages (ED1). A–C) Cor- pus, D–F) distal cauda. A, D) 3 mo, B, E) 18 mo with numerous spermatozoa in the lumen, C, F) 18 mo with occasional sper- matozoa in the lumen. lu, Lumen; it, inter- tubular space; p, principal cells; b, basal cells; c, clear cells; bm, basement mem- brane. Arrows, ED1-positive cells; aster- isks, halo cells. Scale bar, A–F 5 64 mm.

Article Snippet: Mouse anti-rat ED1 (Serotec USA, Washington, DC) was used at a dilution 1/100; this antibody recognizes a cytoplasmic antigen in monocytes and most macrophages.

Techniques: Staining

Journal: Biology of reproduction

Article Title: Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content.

doi: 10.1095/biolreprod61.3.705

Figure Lengend Snippet: FIG. 5. Light micrographs showing sec- tions of the corpus of the epididymis of Brown Norway rats. A, C) Three mo; B, D) 18 mo. Section stained with an antibody for A and B GST Yf and C and D ED1. Clear arrows, basal cells; dark arrows, ED1-positive cells; other labels as in Fig- ure 1. Scale bar, A–D 5 64 mm.

Article Snippet: Mouse anti-rat ED1 (Serotec USA, Washington, DC) was used at a dilution 1/100; this antibody recognizes a cytoplasmic antigen in monocytes and most macrophages.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Multiple Immunostainings with Different Epitope Retrievals—The FOLGAS Protocol

doi: 10.3390/ijms23010223

Figure Lengend Snippet: Secondary antibodies, chromogens, fluorophores and metals. Except DAPI, the fluorophores not directly coupled to antibodies are linked to streptavidin for binding of biotinylated antibodies. Abbreviations: RTU: ready to use, AP: alkaline phosphatase, HRP: horseradish peroxidase. Tb: Terbium, Er: Erbium.

Article Snippet: , 159Tb (conjugated to CD68 clone KP1) , , - , Fluidigm (SKU 201300).

Techniques: Binding Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression.

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: Fig. 2 High SERPINE1 expression in GC cells promotes macrophage M2 polarization. tSNE visualization of nine single-cell clusters partitioned by unsu pervised cluster analysis, SERPINE1 expression of each single-cell, and SERPINE1 expression abundance of different single-cell clusters in the GSE134520 (A–C) and GSE167297 (D–F) datasets. (G) Flow cytometry analysis of the proportion of CD68+CD206+ macrophages in a Transwell co-culture system, with MKN45 and AGS cells overexpressing (oe_SERPINE1) or silencing SERPINE1 (shRNA#3 or sh_SERPINE1#3) in the upper chamber, and THP1 cells treated with PMA in the lower chamber. (H) Immunofluorescence staining of xenograft tumor tissues. Comparison of the proportion of M1 or M2 macrophage infiltra tion. Green indicates F4/80. Red indicates iNOS or Arg1 expression

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68+CD206+ populations by flow cytometry (Accuri C6, BD).

Techniques: Expressing, Flow Cytometry, Co-Culture Assay, shRNA, Immunofluorescence, Staining, Comparison

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression.

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: Fig. 5 SERPINE1-mediated gastric cancer-derived exosomes facilitate the polarization of THP1 cells into M2 macrophages. (A) Schematic representation of the extraction and identification of exosomes and the induction of macrophage polarization. Transmission electron microscopy (B), nanoparticle tracking analysis (C), and western blotting (D) were used to identify the morphology, particle size, and markers of exosomes. (E) Confocal laser scanning microscopy detected Dil-labeled exosomes (red) internalized by DAPI-labeled macrophages (blue). (F–G) Immunofluorescence analysis of the proportion of CD206+ cells in THP1 cells treated with exosomes. (H–I) Flow cytometry analysis of the proportion of CD68+CD206+ cells in THP1 cells treated with exosomes. (J–K) qRT-PCR analysis of M1 markers (iNOS and TNF-α) and M2 markers (TGF-β, IL-10, and Arg-1) in THP1 cells treated with exosomes. (L–N) Transwell migration and invasion assays of GC cells (upper chamber) co-cultured with macrophages (lower chamber) ingesting exosomes

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68+CD206+ populations by flow cytometry (Accuri C6, BD).

Techniques: Derivative Assay, Extraction, Transmission Assay, Electron Microscopy, Western Blot, Confocal Laser Scanning Microscopy, Labeling, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Migration, Cell Culture

Journal: World Journal of Gastroenterology : WJG

Article Title: Protection against hepatic ischemia/reperfusion injury via downregulation of toll-like receptor 2 expression by inhibition of Kupffer cell function

doi: 10.3748/wjg.v11.i28.4423

Figure Lengend Snippet: (PDF) Effect of GdCl3 injection on CD68 ( A) and TLR2 ( B) expression in blockade and non-blockade groups. bP<0.01vs non-blockade group.

Article Snippet: Four micrometers of thick sections were stained with anti-CD68 Ab (Boster Bio.

Techniques: Injection, Expressing

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Estrogen-sensitive activation of SGK1 induces M2 macrophages with anti-inflammatory properties and a Th2 response at the maternal–fetal interface

doi: 10.1186/s12958-023-01102-9

Figure Lengend Snippet: Serum E 2 declines as gestational age increases in early miscarriage and SGK1 is downregulated in decidual macrophages of RPL. A The concentrations of serum E 2 (pg/ml) during the 4 th –12 th week of gestation in a study population with live birth ( n = 448) or miscarriage ( n = 68) in the first trimester. B The variation of serum E 2 (pg/ml) with weeks during early pregnancy in women with a viable fetus or miscarriage. C Decidual tissue sections obtained from first trimester of gestation were double stained with anti-human SGK1 antibody (red) and anti-human CD68 antibody (green) using laser scanning confocal microscopy; nuclear DNA was stained with DAPI (blue). The yellow-orange color in the merged images indicates the colocalization of SGK1 and CD68 + macrophages in decidual tissue, n = 3, at 630 × magnification, scale bar 25 μm. D The ordinate represents the quantification of immunoreactive SGK1 levels/decidual area (μm 2 ) acquired in (C). E The ordinate indicates the quantification of immunoreactive SGK1 staining in decidual CD68 + macrophages obtained from 1C. ** P < 0.01, *** P < 0.001, contrasted to normal pregnancy group. E 2 , estradiol; SGK1, serum-glucocorticoid regulated kinase; RPL, recurrent pregnancy loss; CD68, CD68 molecule; DAPI, 4’,6-diamidino-2-phenylindole

Article Snippet: For labeling, decidual tissue sections were incubated with primary antibodies: mouse monoclonal anti-SGK1 antibodies (1:50, SC-28338, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-CD68 antibodies (1:500, 76437, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C.

Techniques: Staining, Confocal Microscopy

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Estrogen-sensitive activation of SGK1 induces M2 macrophages with anti-inflammatory properties and a Th2 response at the maternal–fetal interface

doi: 10.1186/s12958-023-01102-9

Figure Lengend Snippet: E 2 upregulates SGK1 activity in THP-1 monocyte-derived macrophages via ERβ. A Representative immunofluorescence staining of THP-1 monocyte-derived macrophages before (resting phenotype, top), and after (differentiated phenotype, bottom) PMA incubation. The differentiation of macrophages was confirmed by staining with the antibodies against macrophage-specific CD68 (green) and with DAPI (blue) for nuclei. Final magnification: × 630, scale bar 25 μm. B Western blotting analysis of THP-1 monocyte-derived macrophages treated with E 2 (10 nM) alone, E 2 plus ER antagonist ICI182780 (1 μM), E 2 plus ERα antagonist MPP (1 μM) and E 2 plus ERβ antagonist PHTPP (1 μM) for 24 h. Blots were probed with antibodies to t-SGK1, p-SGK1, and β-actin. C Densitometric quantifications of the arithmetic mean (SEM) ratio of p-SGK1 (left), t-SGK1 (middle), and p/t-SGK1 protein (right) to β-actin in THP-1 monocyte-derived macrophages. Western blotting analysis ( D ) and densitometric quantifications ( E ) of THP-1 monocyte-derived macrophage lysates pretreated with an ESR2 (encoding ERβ)-specific siRNA and non-targeting (NT) siRNA. Blots were probed with antibodies to p-SGK1 (left), t-SGK1 (middle), and p/t SGK1 protein (right) in THP-1 monocyte-derived macrophage lysates. β-actin was the loading control. F Blots were probed with antibodies to ESR2 and β-actin prepared from lysates of cell pretreated with NT siRNA or ESR2 siRNA. Three independent samples were analyzed. Data are expressed as the arithmetic means ± SEM. ** P < 0.01, *** P < 0.001, contrasted with control or medium group; ∆∆∆ P < 0.001, contrasted with E 2 group; ### P < 0.001, contrasted with the E 2 + ICI 182780 or E 2 + NT siRNA group; + + + P < 0.001, contrasted with the E 2 + PHTPP group. E 2 , estradiol; SGK1, serum-glucocorticoid regulated kinase; ER, estrogen receptor; PMA, phorbol 12-myristate 13-acetate; CD68, CD68 molecule; DAPI, 4′,6-diamidino-2-phenylindole; NT, non-targeting; siRNA, small interfering RNA; SEM, Standard Error of the Mean

Article Snippet: For labeling, decidual tissue sections were incubated with primary antibodies: mouse monoclonal anti-SGK1 antibodies (1:50, SC-28338, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-CD68 antibodies (1:500, 76437, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C.

Techniques: Activity Assay, Derivative Assay, Immunofluorescence, Staining, Incubation, Western Blot, Control, Small Interfering RNA

Journal: Oncology Letters

Article Title: Accumulation of stabilin-1 positive macrophages in the early stage of gastric cancer is associated with short cumulative survival

doi: 10.3892/ol.2020.11310

Figure Lengend Snippet: Comparison of number of CD68 + and stabilin-1-positive cells in cancer tissue and remote non-cancerous tissue of primary gastric cancer.

Article Snippet: The following primary antibodies were used: Mouse anti-human CD68 monoclonal antibody (1:50; cat. no. MSK055 KP-1; Zytomed Systems GmbH), rabbit anti-human stabilin-1 polyclonal antibody (2 μg/ml; 1:800; clone number #27606), kindly provided by Professor Julia Kzhyshkowska (Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Germany), goat anti-human CD163 antibody (1:200; cat. no. 1607; R&D Systems Europe, Ltd.) and goat anti-human SPARC polyclonal antibody (1:60; cat. no. AF941; R&D Systems, Inc.).

Techniques: Comparison

Journal: Oncology Letters

Article Title: Accumulation of stabilin-1 positive macrophages in the early stage of gastric cancer is associated with short cumulative survival

doi: 10.3892/ol.2020.11310

Figure Lengend Snippet: Expression of stabilin-1 by CD68 + macrophages in human gastric adenocarcinoma. Stabilin-1 was detected by rabbit anti-human stabilin-1 polyclonal antibody and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (indicated in green). CD68 was detected by mouse anti-human CD68 monoclonal antibody and Cy3-conjugated donkey anti-mouse IgG (indicated in red). Nuclei were stained with DRAQ5 (indicated in blue). (A) Stabilin-1-negative/CD68 + cells in poorly gastric adenocarcinoma. Scale bar, 25 µm. (B) Stabilin-1-negative/CD68 + and stabilin-1-positive/CD68 + cells in moderately differentiated gastric adenocarcinoma. Scale bar, 25 µm. (C) Stabilin-1-positive/CD68 + cells in well differentiated gastric adenocarcinoma. Scale bar, 25 µm. (D) Typical stabilin-1-positive/CD68 + cell in moderately differentiated adenocarcinoma. Scale bar, 10 µm.

Article Snippet: The following primary antibodies were used: Mouse anti-human CD68 monoclonal antibody (1:50; cat. no. MSK055 KP-1; Zytomed Systems GmbH), rabbit anti-human stabilin-1 polyclonal antibody (2 μg/ml; 1:800; clone number #27606), kindly provided by Professor Julia Kzhyshkowska (Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Germany), goat anti-human CD163 antibody (1:200; cat. no. 1607; R&D Systems Europe, Ltd.) and goat anti-human SPARC polyclonal antibody (1:60; cat. no. AF941; R&D Systems, Inc.).

Techniques: Expressing, Staining

Journal: Oncology Letters

Article Title: Accumulation of stabilin-1 positive macrophages in the early stage of gastric cancer is associated with short cumulative survival

doi: 10.3892/ol.2020.11310

Figure Lengend Snippet: Expression of stabilin-1 by CD68 + /CD163 + macrophages in human gastric adenocarcinoma demonstrated by immunofluorescence staining. Stabilin-1 was detected by rabbit anti-human stabilin-1 polyclonal antibody and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (indicated in green). CD163 was detected by goat anti-human CD163 antibody Cy3-conjugated donkey anti-goat IgG (indicated in red). CD68 was detected by mouse anti-human CD68 monoclonal antibody and Alexa Fluor 647-conjugated donkey anti-mouse IgG (indicated in blue). (A) CD68 + /CD163 + /stabilin-1- negative macrophages in gastric adenocarcinoma. Scale bar, 25 µm. (B) CD68 + /CD163 low /stabilin-1-positive macrophages in gastric adenocarcinoma. Scale bar, 25 µm. (C) CD68 + /CD163 low /stabilin-1-positive and CD68 + /CD163 + /stabilin-1-positive cells in gastric adenocarcinoma. Scale bar, 25 µm. (D) Single CD68 + /CD163 + /stabilin-1-positive cell in gastric adenocarcinoma. Scale bar, 2.5 µm.

Article Snippet: The following primary antibodies were used: Mouse anti-human CD68 monoclonal antibody (1:50; cat. no. MSK055 KP-1; Zytomed Systems GmbH), rabbit anti-human stabilin-1 polyclonal antibody (2 μg/ml; 1:800; clone number #27606), kindly provided by Professor Julia Kzhyshkowska (Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Germany), goat anti-human CD163 antibody (1:200; cat. no. 1607; R&D Systems Europe, Ltd.) and goat anti-human SPARC polyclonal antibody (1:60; cat. no. AF941; R&D Systems, Inc.).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Oncology Letters

Article Title: Accumulation of stabilin-1 positive macrophages in the early stage of gastric cancer is associated with short cumulative survival

doi: 10.3892/ol.2020.11310

Figure Lengend Snippet: Association of stabilin-1 and CD68 expression in TNM stage I gastric cancer with cumulative patient survival. Kaplan-Meier analysis and a log rank test were used to compare patient survival with low and high numbers of intratumoral stabilin-1-positive and CD68 + cells. (A) The association of stabilin-1 expression with patient survival. (B) The association of CD68 expression with patient survival. The cutoff value for determining CD68 and stabilin-1 expression as low and high expression was 16.2 cells and 8.0 cells, respectively. TNM, Tumor-Node-Metastasis; Cum, cumulative.

Article Snippet: The following primary antibodies were used: Mouse anti-human CD68 monoclonal antibody (1:50; cat. no. MSK055 KP-1; Zytomed Systems GmbH), rabbit anti-human stabilin-1 polyclonal antibody (2 μg/ml; 1:800; clone number #27606), kindly provided by Professor Julia Kzhyshkowska (Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Germany), goat anti-human CD163 antibody (1:200; cat. no. 1607; R&D Systems Europe, Ltd.) and goat anti-human SPARC polyclonal antibody (1:60; cat. no. AF941; R&D Systems, Inc.).

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Fibrinogen-like protein 2 prothrombinase may contribute to the progression of inflammatory bowel disease by mediating immune coagulation

doi:

Figure Lengend Snippet: Immunohistochemical staining of various indicators. TNF-α in IBD mice and patients (B and F, ×400), and normal controls (A and E ×400). FGL2 in IBD mice and patients (C and J, ×400), and normal controls (D ×200 and I ×400). Fibrin in normal control and IBD patients (H ×200 and G ×400). CD68 in normal control and IBD patients (L and K, ×400).

Article Snippet: Immunoperoxidase staining of macrophages A CD68 Ab (ProteinTech, at a dilution of 1:100) was used to detect macrophages using the similar methodology described above.

Techniques: Immunohistochemical staining, Staining, Control