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a, Representative images of Cxcl15 fluorescent detection (murine homologue of IL-8) in Myc-Cap tumors. Tumors were harvested when volumes reached ∼500mm (CS group), 7 days after androgen-deprivation (ADT), or at the time of castration-resistance (CR) and hybridized with CF568-labeled probe sets (white) to Cxcl15 , <t>CF640-labeled</t> <t>anti-PanCK</t> antibody (red), and CF488-labeled anti-CD45 antibody (green). Nuclei counterstained with DAPI (blue). Repeated × 3. b, Gene and protein expression of Cxcl15 in MCRedAL cells of indicated tumor samples by qRT-PCR and ELISA, respectively (n = 3 per group, repeated × 2). c, qRT-PCR quantification of IL-8 in human AR positive castration-sensitive cells (CS: LNCaP, LAPC4, and VCaP) and their castration-resistant counterparts (CR: LNCaP-abl, LAPC4-CR, and VCaP-CR), replicate numbers as in b . d, IL-8 protein expression in the isogenic cell pairs from c quantified by ELISA, replicate numbers as in c . e, Representative images of Cxcl15 fluorescent detection in benign murine prostate tissue samples from castration-sensitive (CS), androgen-deprivation treated (ADT), and ADT-treated mice that received testosterone repletion (ADT + T). Tissue sections hybridized with CF568-labeled probe sets (white) to Cxcl15 , and CF640-labeled anti-PanCK antibody (red). Nuclei were counterstained with DAPI (blue). Repeated × 3. f, qRT-PCR analysis of Cxcl15 expression in prostate luminal epithelial cells from indicated treatment groups (n = 3 per group). Prostate luminal epithelial cells were isolated based on their GFP + CD49f int CD24 + CD45 - F4/80 - CD11b - expression by flow sorting into Trizol LS. g, Expression of IL-8 in human prostate epithelial cells micro-dissected from patients in a clinical trial ( NCT00161486 ) receiving placebo, androgen-deprivation treatment (ADT), or ADT plus testosterone repletion (ADT + T). Z-score values of microarray transcripts from benign prostate biopsies were normalized to placebo samples (n = 4 per group; GSE8466). h, Expression of IL-8 in human prostate cancer epithelial cells micro-dissected from untreated or ADT-treated ( NCT01696877 ; n = 8 per group) patients as determined by qRT-PCR. RISH images are at 60X magnification; scale bar = 100 μm. Gene expression levels were normalized to the mean ΔCT level in samples from CS, untreated or placebo groups. For b-g , unpaired t-tests were performed; for h a Mann-Whitney U test was used due to the non-normal data distribution observed. p -values ≤ 0.05 (*) and 0.01 (**); p -values ≥ 0.05 (ns) shown. The range in box and whiskers plots shows min and max values such that all data are included.
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a, Representative images of Cxcl15 fluorescent detection (murine homologue of IL-8) in Myc-Cap tumors. Tumors were harvested when volumes reached ∼500mm (CS group), 7 days after androgen-deprivation (ADT), or at the time of castration-resistance (CR) and hybridized with CF568-labeled probe sets (white) to Cxcl15 , CF640-labeled anti-PanCK antibody (red), and CF488-labeled anti-CD45 antibody (green). Nuclei counterstained with DAPI (blue). Repeated × 3. b, Gene and protein expression of Cxcl15 in MCRedAL cells of indicated tumor samples by qRT-PCR and ELISA, respectively (n = 3 per group, repeated × 2). c, qRT-PCR quantification of IL-8 in human AR positive castration-sensitive cells (CS: LNCaP, LAPC4, and VCaP) and their castration-resistant counterparts (CR: LNCaP-abl, LAPC4-CR, and VCaP-CR), replicate numbers as in b . d, IL-8 protein expression in the isogenic cell pairs from c quantified by ELISA, replicate numbers as in c . e, Representative images of Cxcl15 fluorescent detection in benign murine prostate tissue samples from castration-sensitive (CS), androgen-deprivation treated (ADT), and ADT-treated mice that received testosterone repletion (ADT + T). Tissue sections hybridized with CF568-labeled probe sets (white) to Cxcl15 , and CF640-labeled anti-PanCK antibody (red). Nuclei were counterstained with DAPI (blue). Repeated × 3. f, qRT-PCR analysis of Cxcl15 expression in prostate luminal epithelial cells from indicated treatment groups (n = 3 per group). Prostate luminal epithelial cells were isolated based on their GFP + CD49f int CD24 + CD45 - F4/80 - CD11b - expression by flow sorting into Trizol LS. g, Expression of IL-8 in human prostate epithelial cells micro-dissected from patients in a clinical trial ( NCT00161486 ) receiving placebo, androgen-deprivation treatment (ADT), or ADT plus testosterone repletion (ADT + T). Z-score values of microarray transcripts from benign prostate biopsies were normalized to placebo samples (n = 4 per group; GSE8466). h, Expression of IL-8 in human prostate cancer epithelial cells micro-dissected from untreated or ADT-treated ( NCT01696877 ; n = 8 per group) patients as determined by qRT-PCR. RISH images are at 60X magnification; scale bar = 100 μm. Gene expression levels were normalized to the mean ΔCT level in samples from CS, untreated or placebo groups. For b-g , unpaired t-tests were performed; for h a Mann-Whitney U test was used due to the non-normal data distribution observed. p -values ≤ 0.05 (*) and 0.01 (**); p -values ≥ 0.05 (ns) shown. The range in box and whiskers plots shows min and max values such that all data are included.

Journal: bioRxiv

Article Title: Castration-mediated IL-8 Promotes Myeloid Infiltration and Prostate Cancer Progression

doi: 10.1101/651083

Figure Lengend Snippet: a, Representative images of Cxcl15 fluorescent detection (murine homologue of IL-8) in Myc-Cap tumors. Tumors were harvested when volumes reached ∼500mm (CS group), 7 days after androgen-deprivation (ADT), or at the time of castration-resistance (CR) and hybridized with CF568-labeled probe sets (white) to Cxcl15 , CF640-labeled anti-PanCK antibody (red), and CF488-labeled anti-CD45 antibody (green). Nuclei counterstained with DAPI (blue). Repeated × 3. b, Gene and protein expression of Cxcl15 in MCRedAL cells of indicated tumor samples by qRT-PCR and ELISA, respectively (n = 3 per group, repeated × 2). c, qRT-PCR quantification of IL-8 in human AR positive castration-sensitive cells (CS: LNCaP, LAPC4, and VCaP) and their castration-resistant counterparts (CR: LNCaP-abl, LAPC4-CR, and VCaP-CR), replicate numbers as in b . d, IL-8 protein expression in the isogenic cell pairs from c quantified by ELISA, replicate numbers as in c . e, Representative images of Cxcl15 fluorescent detection in benign murine prostate tissue samples from castration-sensitive (CS), androgen-deprivation treated (ADT), and ADT-treated mice that received testosterone repletion (ADT + T). Tissue sections hybridized with CF568-labeled probe sets (white) to Cxcl15 , and CF640-labeled anti-PanCK antibody (red). Nuclei were counterstained with DAPI (blue). Repeated × 3. f, qRT-PCR analysis of Cxcl15 expression in prostate luminal epithelial cells from indicated treatment groups (n = 3 per group). Prostate luminal epithelial cells were isolated based on their GFP + CD49f int CD24 + CD45 - F4/80 - CD11b - expression by flow sorting into Trizol LS. g, Expression of IL-8 in human prostate epithelial cells micro-dissected from patients in a clinical trial ( NCT00161486 ) receiving placebo, androgen-deprivation treatment (ADT), or ADT plus testosterone repletion (ADT + T). Z-score values of microarray transcripts from benign prostate biopsies were normalized to placebo samples (n = 4 per group; GSE8466). h, Expression of IL-8 in human prostate cancer epithelial cells micro-dissected from untreated or ADT-treated ( NCT01696877 ; n = 8 per group) patients as determined by qRT-PCR. RISH images are at 60X magnification; scale bar = 100 μm. Gene expression levels were normalized to the mean ΔCT level in samples from CS, untreated or placebo groups. For b-g , unpaired t-tests were performed; for h a Mann-Whitney U test was used due to the non-normal data distribution observed. p -values ≤ 0.05 (*) and 0.01 (**); p -values ≥ 0.05 (ns) shown. The range in box and whiskers plots shows min and max values such that all data are included.

Article Snippet: Primary antibody for PanCK was diluted 1/400 in renaissance background reducing diluent (Biocare Medical) and incubated overnight at 4 °C.

Techniques: Labeling, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, Microarray, MANN-WHITNEY