rabbit anti mouse cd68 igg  (Bio-Rad)

Journal: Frontiers in Immunology

Article Title: Magnolol Attenuates Cisplatin-Induced Muscle Wasting by M2c Macrophage Activation

doi: 10.3389/fimmu.2020.00077

Figure Lengend Snippet: Magnolol increased macrophages and IGF-1 expression in TA muscle. Cross-sections and total mRNA or protein were obtained from TA muscles in the control (con), magnolol (mag), cisplatin (cis), or cis+mag groups. (A) Immunofluorescence staining for anti-myosin heavy chain (MYHC; red) and CD68 macrophages (green) in TA muscle tissues (merged yellow pixels: upper panel, green channel only: lower panel). 4'6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei (blue). White arrows indicate the CD68-stained cells near DAPI. Magnification: ×40, Scale bar: 50 μm. (B) Count of CD68 + macrophages (white arrows in A ) per image area (mm 2 ). At least 5 random fields per section were analyzed. The events that do not contain DAPI staining were not counted. (C) IGF-1 mRNA expression measured by qPCR in TA muscle tissues. GAPDH was used as reference and the level of the genes was normalized to 1. (D) Quantification of IGF-1 protein expression by ELISA in TA muscle tissues. (E) Immunofluorescence staining showing IGF-1 (red) and CD68 + macrophages (green) in TA muscle. Magnification: ×40, Scale bar: 50 μm. CD68 + macrophages with IGF-1 expression were enlarged in red box. Magnification: ×40, Scale bar: 20 μm. Data are representative of three individual experiments, and all graphs are expressed as the mean ± SEM of 5 mice. *** P < 0.001 vs. con, # P < 0.05 vs. mag, and $ P < 0.05; $$ P < 0.01 vs. cis based on the one-way ANOVA Tukey's test. The letters for no significance were not shown.

Article Snippet: CD163 expression on CD68 + macrophages were detected with rabbit anti-mouse CD68 (1:1,000; Santa Cruz, CA, USA), rat anti-CD163 (1:1,000; 14-1631-82; e-bioscience, CA, USA), Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11008; Invitrogen), and Alexa Fluor 594-conjugated goat anti-rat IgG (A11007; Invitrogen).

Techniques: Expressing, Muscles, Control, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Magnolol Attenuates Cisplatin-Induced Muscle Wasting by M2c Macrophage Activation

doi: 10.3389/fimmu.2020.00077

Figure Lengend Snippet: Magnolol attenuated the M1/M2 imbalance caused by cisplatin damage. Phenotypic changes in macrophages by magnolol treatment were analyzed in TA muscle tissue using immunostaining and flow cytometry. TA muscles were obtained from control (con), magnolol (mag) cisplatin (cis), or cis+mag mice at the end of the experiment (day 42). (A) Macrophage phenotyping using immunofluorescence staining with the pan-macrophage marker CD68 (green) and M2 regenerative macrophage specific marker CD163 (red). Magnification: ×40, Scale bar: 50 μm. Merged macrophages were enlarged in red box, Scale bar: 20 μm. (B) CD163 + /CD163 − ratio calculated by dividing the numbers of CD68 + CD163 + merged yellow pixel by those of CD68 + CD163 − green pixel. (C) Flow cytometry analysis of CD86 + CD206 − M1, CD86 + CD206 + M2b, CD86-CD206 + CD163 − M2a, and CD86 − CD206 + CD163 + M2c macrophages. Representative contour plots of macrophages characterized by CD86 and CD206 gated on CD45 + CD11b + F4/80 + subsets (upper panel) and M2a or M2c macrophages characterized by CD163 and CD206 gated on CD86 − CD206 + subsets (lower panel). (D) Bar graphs showing mean% of M1, M2b, and M2a/c macrophages in CD11b + F4/80 + cells and (E) mean% of M2c macrophages in CD11b + F4/80 + cells. (F) M1/M2c ratio calculated based on the number of M1 and M2c macrophages in CD45 + CD11b + F4/80 + gated cells. Data represent mean ± SEM of 5 mice, and all data are representative from three individual experiments. * P < 0.05; ** P < 0.01 compared to con, # P < 0.05; * P < 0.05 compared to mag, and $ P < 0.05; $$ P < 0.01; $$$ P < 0.001 compared to cis using Turkey's test. The letters for no significance were not shown.

Article Snippet: CD163 expression on CD68 + macrophages were detected with rabbit anti-mouse CD68 (1:1,000; Santa Cruz, CA, USA), rat anti-CD163 (1:1,000; 14-1631-82; e-bioscience, CA, USA), Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11008; Invitrogen), and Alexa Fluor 594-conjugated goat anti-rat IgG (A11007; Invitrogen).

Techniques: Immunostaining, Flow Cytometry, Muscles, Control, Immunofluorescence, Staining, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Phytosomal curcumin causes natural killer cell-dependent repolarization of glioblastoma (GBM) tumor-associated microglia/macrophages and elimination of GBM and GBM stem cells

doi: 10.1186/s13046-018-0792-5

Figure Lengend Snippet: NK1.1Ab treatment partially reverses the CCP-evoked elimination of CD133(+) GBM stem cells and CD68 high GBM cells. The GBM tumor in each mouse was cut into two parts and processed for IHC and Flow Cytometry, respectively. a - b IHC: the Vehicle-treated mouse GBM sections harbored a significant number of CD133(+) GBM stem cells (red) ( a first row). Compared to the Vehicle-treated, the CD133(+) fluorescence showed a 72% decrease in the CCP-treated ( * p = 6.5 × 10 − 3 CCP versus Vehicle) ( a , second row and b ) and a 41% decrease in the CCP + NK1.1-treated mice ( a third row and b ) (Δ p < 0.02, CCP + NK1.1 versus CCP; ** p = 0.03, CCP + NK1.1 versus Vehicle). (Scale bar: 47.62 μm). c - f Flow Cytometry: compared to the Vehicle-treated, the CD133(+) cells ( c , top, red circle) were suppressed by 81% in the CCP-treated ( c middle and CD133 IF shown in d ) (* p < 0.05, CCP versus Vehicle) and by 32% in the CCP + NK1.1 mice ( c , bottom, and d ) (Δ p < 0.03, CCP + NK1.1 versus CCP; ** p = 3.0 × 10 − 3 , CCP + NK1.1 versus Vehicle). e The fluorescence profiles of the CD133(+) cells in the Vehicle, CCP, and CCP + NK1.1 samples. f - h The GBM cells were also probed for CD68, and active-caspase-3 (Act. Cspse 3). f i In the Vehicle-treated, two segregated populations of CD68(+) but Cspse3(−) cells were identified (Lower Right, LR). The larger population (shown within the blue circle) was CD68 high (presumably GBM cells) whereas a smaller population (shown within the green circle) was CD68 low (expected to be the TAM) [ , , ]. A very small population of CD68(+), Act. Cspse3(+) (double positive) cells was identified in the upper right (UR) quadrant (within the red ellipse). f ii In the CCP-treated, CD68 high cells showed an 1187% increase in Act. Cspse 3 IF (red ellipse in the UR quadrant) with respect to Vehicle, along with the virtual disappearance of the CD68 high but Cspse3(−) cells in the blue circle in the LR quadrant (* p = 0.036, CCP versus Vehicle) ( f ii and g ). The Act. Cspse 3 IF increased by 343% in the CCP + NK1.1 samples, along with the reappearance of some CD68 hgh but Act. Cspse 3(−) cells in the blue circle in the LR quadrant ( f iii and g ) (Δ p < 0.04, CCP versus CCP + NK1.1; ** p = 3.1 × 10 − 3 , CCP + NK1.1 versus Vehicle). ( f iv ) Fluorescence profiles for Act.Cspse 3. The CD68 fluorescence in CD68 low cells (TAM) was not significantly different in the three groups ( h ). The graphs represent mean ± S.D. obtained from mice treated with Vehicle ( n = 4), CCP ( n = 4), and CCP + NK1.1 ( n = 3)

Article Snippet: Antibodies against Iba1 (C20) (1:50), iNOS (rabbit IgG) (NOS2 sc-651) (1:100), ARG1 (rabbit IgG) (sc-20,150) (1:100), NKp46 (1:100), IL12p40 (1:50), IL10 (1:50), CD68 (H-255) (Rabbit IgG) (sc-9139) (1:50), Active-Caspase3 (Asp175) (Rabbit IgG) (CST #9661) (1:100), and CD133 (1:50) were used for staining.

Techniques: Flow Cytometry, Fluorescence

Journal: Molecular Cancer

Article Title: Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7

doi: 10.1186/1476-4598-13-206

Figure Lengend Snippet: Let-7d expression in RCC cell line and tumor samples, and correlation with tumor stromal cells. (A) Real-time RT-PCR analysis of relative let-7d expression in human cell lines. The data represent the average ± SD of three independent RT-PCR results. (B) The relative expression of let-7d in 80 RCC tissues and matched adjacent normal tissues were analyzed by real-time RT-PCR. (C, D) Real-time RT-PCR analysis of relative let-7d expression in 80 RCC tissues in different T stages (C) and tumor grades (D) . Horizontal lines in (B–D) represent the mean values of relative let-7d expression for each series of samples. Note that (B–D) were generated from the same data as Table . (E) A linear regression and correlation among relative let-7d expression in a log scale vs. CD68 positive cell counts in five independent fields under × 20 objective lens is shown with r (Spearman) and P-values indicated.

Article Snippet: Paraffin embedded tissues were analyzed using immunohistochemical staining [ ] with the following primary antibodies: anti-CD68 antibody (DAKO, Carpinteria, CA), anti-α-SMA antibody (DAKO, Carpinteria, CA), anti-CCL7 antibody (Gen Way Biotech, San Diego, CA), anti-COL3A1 antibody (Bioss, Beijing, China), anti-FOXP3 antibody (Biolegend, San Diego, CA) and rabbit anti-Mouse CD68 antibody (Bioss, Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Generated

Journal: Molecular Cancer

Article Title: Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7

doi: 10.1186/1476-4598-13-206

Figure Lengend Snippet: Overexpression of let-7d in vivo. (A, B) Growth curves and average net weights of xenografts formed by 786O-v (n = 5) and 786O-let-7d cells (n = 5). (C, D) Representative micrographs and quantitative data of metastatic colonies in mice lung. DiI-positive lung metastatic colonies were photographed and counted under a laser confocal microscope. (E) Real-time PCR quantification of relative Alu-sequence expression in mice lung. (F) Quantitative data of the mean CD68 positive cells per five fields in each group. (G) Representative picture of IHC staining of CD68 positive cells. Original magnification: ×200. Data in (A–F) represent the mean ± SD of five mice per group. P values were obtained by the two-tailed Student’s t-test. * P < 0.05. (H) HE staining of the patient tumor and its corresponding derived xenograft (2nd passage). Original magnification: ×200. (I) Real-time RT-PCR analysis of let-7d expression in human RCC tissue, paired normal adjacent tissue, and corresponding PDX tumor tissues 1 week after intratumoral injection of let-7d mimics or control RNA. (J, K) Growth curves and average net weights of xenografts injected with let-7d mimics (n = 7), control RNA (n = 7) or PBS control (n = 7). (L, M) Representative pictures and quantitative data of metastatic colonies in mice lung. (N) Real-time PCR quantification of relative human-specific Alu-sequence expression in mice lung. The values in (J–N) are presented as the mean ± SD of seven mice. P values were obtained by one-way ANOVA. ** P < 0.01 (let-7d mimics vs. PBS or control RNA).

Article Snippet: Paraffin embedded tissues were analyzed using immunohistochemical staining [ ] with the following primary antibodies: anti-CD68 antibody (DAKO, Carpinteria, CA), anti-α-SMA antibody (DAKO, Carpinteria, CA), anti-CCL7 antibody (Gen Way Biotech, San Diego, CA), anti-COL3A1 antibody (Bioss, Beijing, China), anti-FOXP3 antibody (Biolegend, San Diego, CA) and rabbit anti-Mouse CD68 antibody (Bioss, Beijing, China).

Techniques: Over Expression, In Vivo, Microscopy, Real-time Polymerase Chain Reaction, Sequencing, Expressing, Immunohistochemistry, Two Tailed Test, Staining, Derivative Assay, Quantitative RT-PCR, Injection