Journal: Scientific Reports

Article Title: RhoA and vigilin are candidates for immunohistochemical markers for epithelioid malignant mesothelioma

doi: 10.1038/s41598-022-20334-0

Figure Lengend Snippet: Immunohistochemistry of tissue samples from one representative case of malignant mesothelioma. ( a ) RhoA, ( b ) vigilin, ( c ) D2-40, ( d ) calretinin, ( e ) BAP1, ( f ) WT1. Scale Bars show 50 μm. Original magnification is × 200 for all except the inset photo in ( c ) (× 400).

Article Snippet: To confirm the diagnosis of MM, additional IHC was performed using a Ventana BenchMark AutoStainer (Ventana Medical Systems, Tucson, AZ) with a primary antibody against BAP1 (clone C4, rabbit monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA), WT1 (clone 6F-H2, Mouse, Dako M3561, Agilent, Santa Clara, California), and calretinin (rabbit polyclonal, Invitrogen 08-1211, Waltham, MA).

Techniques: Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: The Detection of Immunity against WT1 and SMAD4 P130L of EpCAM + Cancer Cells in Malignant Pleural Effusion

doi: 10.3390/ijms232012177

Figure Lengend Snippet: WT1 expression on EpCAM + cancer cells and detection of memory subsets of WT1-CTLs in MPE samples. ( A ) Expression of WT1 on EpCAM positive or negative cells sorted from whole cells in MPE 1st . PC, phase contrast; WT1 cells were stained with anti-WT1 antibody, followed by Alexa Fluor® Plus 488 secondary antibody; DNA, DAPI staining. The white bar indicates 100 μm. ( B ) The ratio of WT-CTLs is defined as WT1 tetramer + CD8 + T cells to MPE samples 1st or MPE 2nd (upper panel). The memory subsets of WT1-CTLs during disease progression (middle panel). CD62L + CD45RO + : central memory T cells (T CM ); CD62L + CD45RO − : naïve T cells (T N ); CD62L − CD45RO + : effector memory T cells (T EM ), CD62L − CD45RO − : terminal effector T cells (T TE ). The function of WT1-CTLs in MPE was evaluated by ELISpot assay for IFN-γ secretion under WT1 235 peptide stimulation (lower panel). The mean number of spots of WT1 235 peptide-specific was shown. The error bar indicated the mean and standard deviation. Unpaired t -test, * p < 0.05.

Article Snippet: Subsequently, 1 × 10 5 cells were fixed with a 10% formalin neutral buffer solution (Wako Pure Chemicals Ltd., Osaka, Japan) for 30 min and then permeabilized with a 0.1% Triton X-100 (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) solution in PBS at room temperature for 5 min and blocked with UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. After blocking, the cells were incubated with a mouse monoclonal primary anti-WT1 antibody (1:100, clone 6F-H2, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 16 h. Subsequently, a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor plus 488 (1:200, Thermo Fisher Scientific) was added at room temperature for 30 min.

Techniques: Expressing, Staining, Biomarker Discovery, Enzyme-linked Immunospot, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Detection of Immunity against WT1 and SMAD4 P130L of EpCAM + Cancer Cells in Malignant Pleural Effusion

doi: 10.3390/ijms232012177

Figure Lengend Snippet: Detection of CD8 + T cell response to HLA-A*11:01 restricted SMAD4 P130L neoantigen in MPE. ( A ) WT1 tetramer-CD8 + T cells sorted from MPE 1st were expanded with HPL-IFN-DCs containing each predicted neoantigen peptide for SMAD4 P130L . For ELISpot assay to detect IFN-γ production after in vitro expansion, these cells were stimulated using each SMAD4 P130L peptide. Reactivity was shown in the box plot (per each well in 96 plates). DMSO was used as a negative control. A Wilcoxon signed-rank test was performed, * p < 0.05. ( B ) CD8 + T cells purified from in vitro expanded cells with HPL-IFN-DCs contained SMAD4-Neo1 peptides in A were stimulated by HEV0271 cells (HLA-A*11:01 homozygous) containing SMAD4-Neo1 (SVCVNLYH) or SMAD4-WT1 (SVCVNPYH). The reactivity to IFN-γ was evaluated by ELISpot assay. The error bar indicated the mean and standard deviation. Dunnett’s test, * p < 0.05.

Article Snippet: Subsequently, 1 × 10 5 cells were fixed with a 10% formalin neutral buffer solution (Wako Pure Chemicals Ltd., Osaka, Japan) for 30 min and then permeabilized with a 0.1% Triton X-100 (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) solution in PBS at room temperature for 5 min and blocked with UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. After blocking, the cells were incubated with a mouse monoclonal primary anti-WT1 antibody (1:100, clone 6F-H2, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 16 h. Subsequently, a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor plus 488 (1:200, Thermo Fisher Scientific) was added at room temperature for 30 min.

Techniques: Enzyme-linked Immunospot, In Vitro, Negative Control, Purification, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Detection of Immunity against WT1 and SMAD4 P130L of EpCAM + Cancer Cells in Malignant Pleural Effusion

doi: 10.3390/ijms232012177

Figure Lengend Snippet: Memory subsets of WT1 tetramer − CD8 + T cells and detection of SMAD4 P130L -specific CD8 + T cell expanded from MPE samples. ( A ) WT1 tetramer − CD8 + T cells in MPE 1st or MPE 2nd were stained with antibodies against CD62L and CD45RO for memory T cell subsets. ( B ) WT1 tetramer − CD8 + T cells were sorted from MPE 1st or MPE 2nd , expanding in vitro with HPL-IFN-DCs containing SMAD4-Neo1 peptide. These cells were stimulated with SMAD4-Neo1 or DMSO to detect IFN-γ-producing cells by ELISpot assay. The ratio of IFN-γ spots of SMAD4-Neo1 to control was shown in the box plot (per each well in 96 plates). Mann–Whitney U test, * p < 0.05.

Article Snippet: Subsequently, 1 × 10 5 cells were fixed with a 10% formalin neutral buffer solution (Wako Pure Chemicals Ltd., Osaka, Japan) for 30 min and then permeabilized with a 0.1% Triton X-100 (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) solution in PBS at room temperature for 5 min and blocked with UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. After blocking, the cells were incubated with a mouse monoclonal primary anti-WT1 antibody (1:100, clone 6F-H2, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 16 h. Subsequently, a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor plus 488 (1:200, Thermo Fisher Scientific) was added at room temperature for 30 min.

Techniques: Staining, In Vitro, Enzyme-linked Immunospot, Control, MANN-WHITNEY

Journal: Cell reports

Article Title: A molecular atlas of the human postmenopausal fallopian tube and ovary from single-cell RNA and ATAC sequencing

doi: 10.1016/j.celrep.2022.111838

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse WT1 Clone 6F-H2 , Thermo Fisher Scientific , Cat#MA1–46028; RRID:AB_962464.

Techniques: Recombinant, Saline, RNAscope, Multiplex Assay, Diagnostic Assay, Software, Gene Expression, Microscopy, Real-time Polymerase Chain Reaction

Journal: Pakistan Journal of Medical Sciences

Article Title: Differentiation of CD117 + Amniotic Fluid Stem Cells towards Nephron Progenitors

doi: 10.12669/pjms.38.6.4887

Figure Lengend Snippet: Characterization of NPCs colonies for WT1 by immunofluorescence staining as performed on three different amniotic fluid derived NPCs. This figure shows immunofluorescence analysis for positive nuclear marker WT1 (a) Bright field image of NPCs at 40x showed irregular with ill-defined borders having tightly packed cells for the three different amniotic fluid derived NPCs. (b) Counter DAPI nuclear stain showed blue color of Nucleus. (c) Characteristic of NPCs, WT1 is shown in nuclear compartment of cells.

Article Snippet: The amniotic fluid samples were washed thoroughly with Phosphate Buffered Saline then fixed at 1:1 acetone: methanol for twenty minutes at 25°C, after washing with Buffer Saline containing tween 20 (PBST) three times Triton X-100 was use for permeabilization of cell membrane then again washed with PBST, it was clogged by bovine serum albumin (BSA) 1% plus PBS (without Calcium &Magnesium) for 30 minutes at Room Temperature and incubated with primary monoclonal unconjugated antibody WT1 (WT1/857 + 6F-H2 clone, Novus Biologicals, Biotechne, USA) as positive and CD117 (CD117 APC, Thermofisher, 104D2) as a negative control for an hour.

Techniques: Immunofluorescence, Staining, Derivative Assay, Marker

Journal: Cancer Science

Article Title: Sensitive immunohistochemical detection of WT1 protein in tumors with anti‐WT1 antibody against WT1 235 peptide

doi: 10.1111/j.1349-7006.2010.01522.x

Figure Lengend Snippet: Reactivity of WT1‐R antibody against 9‐mer peptide region of Wilms’ tumor 1 (WT1) protein targeted by WT1 vaccine. (A) Dilution curves of WT1‐R. Plates coated with WT1‐GST or GST protein were incubated with serial dilutions of WT1‐R antibody and the binding of WT1‐R antibody to plates was determined. (B) Results of competitive ELISA. Serially diluted WT1 peptide was incubated with or without 3 mg/mL of WT1‐R antibody, and this mixture was then added to GST‐WT1‐coated plates. (C,D) Western blotting analysis with GST or WT1‐R antibody. Proteins were immunoprecipitated with the antibodies indicated above before electrophoresis.

Article Snippet: In addition, mouse monoclonal antibodies to WT1 (clone 6F‐H2; Dako Cytomation, Carpinteria, CA, USA) and to glutathione S ‐transferase (GST; clone 40B3; BioPortfolio, Dorset, UK) were also used for comparison.

Techniques: Wilms Tumor Assay, Incubation, Binding Assay, Competitive ELISA, Western Blot, Immunoprecipitation, Electrophoresis

Journal: Cancer Science

Article Title: Sensitive immunohistochemical detection of WT1 protein in tumors with anti‐WT1 antibody against WT1 235 peptide

doi: 10.1111/j.1349-7006.2010.01522.x

Figure Lengend Snippet: Comparison between immunohistochemical staining with WT1‐R and real‐time quantitative PCR (RT‐PCR) to detect Wilms’ tumor 1 (WT1) expression in five cell lines (Daudi, PC‐14, K562, HT1080, and SW480). RT‐PCR results are shown beneath the photographs. Magnification, ×400.

Article Snippet: In addition, mouse monoclonal antibodies to WT1 (clone 6F‐H2; Dako Cytomation, Carpinteria, CA, USA) and to glutathione S ‐transferase (GST; clone 40B3; BioPortfolio, Dorset, UK) were also used for comparison.

Techniques: Comparison, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Wilms Tumor Assay, Expressing

Journal: Cancer Science

Article Title: Sensitive immunohistochemical detection of WT1 protein in tumors with anti‐WT1 antibody against WT1 235 peptide

doi: 10.1111/j.1349-7006.2010.01522.x

Figure Lengend Snippet: Immunohistochemical analysis of normal gastric mucosa (A) and breast cancer tissue samples from four different patients (B). In (B), the upper and lower photographs show the results of immunohistochemical staining with 6F‐H2 and with WT1‐R, respectively. Magnification, ×120.

Article Snippet: In addition, mouse monoclonal antibodies to WT1 (clone 6F‐H2; Dako Cytomation, Carpinteria, CA, USA) and to glutathione S ‐transferase (GST; clone 40B3; BioPortfolio, Dorset, UK) were also used for comparison.

Techniques: Immunohistochemical staining, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Maintenance of WT1 expression in tumor cells is associated with a good prognosis in malignant glioma patients treated with WT1 peptide vaccine immunotherapy

doi: 10.1007/s00262-021-02954-z

Figure Lengend Snippet: Level of WT1 (a), TGFβ (b), and IL-10 (c) expression was classified as follows: 0, negative staining; 1, slightly increased staining in some tumors cells compared to that in normal glial cells; 2, staining of intermediate intensity in some tumor cells; 3, strong staining in some tumor cells and intermediate staining in almost all tumor cells; and 4, greatly increased staining in almost all tumor cells compared to that in normal glial cells. Expression of PD-L1 on tumor cells was graded as follows: 0, absence of staining; 1, up to 25% of cells stained; 2, 25–50% of cells stained; and 4, more than 50% of cells stained. Immunohistochemistry of tumor infiltrating lymphocytes (TILs). CD8+, CD4+, and PD-1+ TILs were present diffusely throughout the tumor tissue in most positive cases

Article Snippet: The sections were then incubated overnight at 4 °C with anti-human WT1 mouse monoclonal antibody 6F-H2 (DAKO, Carpinteria, CA, USA; diluted 1:50), anti-human Transforming growth factor-β (TGF-β) rabbit polyclonal antibody (Abcam, Cambridge, MA, USA; diluted 1:200), anti–human Ki-67 mouse monoclonal antibody MIB-1 (DAKO; diluted 1:40), anti-human HLA class I-ABC mouse monoclonal antibody EMR8-5 (Hokudo, Sapporo, Japan; diluted 1:100), anti-human programmed death-ligand 1 (PD-L1) rabbit monoclonal antibody ab205921 clone 28–8, (Abcam; diluted 1:100), anti-human CD3 mouse monoclonal antibody F7.2.38 (DAKO; diluted 1:5), anti–human CD4 mouse monoclonal antibody 4B12 (DAKO; diluted 1:80), anti–human CD8 mouse monoclonal antibody C8/14B (Nichirei Biosciences, Tokyo, Japan; undiluted), anti-human CD79α mouse monoclonal antibody JCB117 (DAKO; diluted 1:200), anti-human PD-1 mouse monoclonal antibody clone NAT105 (Abcam; diluted 1:100), anti-human Foxp3 mouse monoclonal antibody 236A/E7 (Abcam; diluted 1:100), or anti IDH1­R132H mouse monoclonal Antibody (FUJIFILM Wako Chemicals, Osaka, Japan; diluted 1:100).

Techniques: Expressing, Negative Staining, Staining, Immunohistochemistry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Maintenance of WT1 expression in tumor cells is associated with a good prognosis in malignant glioma patients treated with WT1 peptide vaccine immunotherapy

doi: 10.1007/s00262-021-02954-z

Figure Lengend Snippet: Patient characteristics and clinical information

Article Snippet: The sections were then incubated overnight at 4 °C with anti-human WT1 mouse monoclonal antibody 6F-H2 (DAKO, Carpinteria, CA, USA; diluted 1:50), anti-human Transforming growth factor-β (TGF-β) rabbit polyclonal antibody (Abcam, Cambridge, MA, USA; diluted 1:200), anti–human Ki-67 mouse monoclonal antibody MIB-1 (DAKO; diluted 1:40), anti-human HLA class I-ABC mouse monoclonal antibody EMR8-5 (Hokudo, Sapporo, Japan; diluted 1:100), anti-human programmed death-ligand 1 (PD-L1) rabbit monoclonal antibody ab205921 clone 28–8, (Abcam; diluted 1:100), anti-human CD3 mouse monoclonal antibody F7.2.38 (DAKO; diluted 1:5), anti–human CD4 mouse monoclonal antibody 4B12 (DAKO; diluted 1:80), anti–human CD8 mouse monoclonal antibody C8/14B (Nichirei Biosciences, Tokyo, Japan; undiluted), anti-human CD79α mouse monoclonal antibody JCB117 (DAKO; diluted 1:200), anti-human PD-1 mouse monoclonal antibody clone NAT105 (Abcam; diluted 1:100), anti-human Foxp3 mouse monoclonal antibody 236A/E7 (Abcam; diluted 1:100), or anti IDH1­R132H mouse monoclonal Antibody (FUJIFILM Wako Chemicals, Osaka, Japan; diluted 1:100).

Techniques: Mutagenesis

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Maintenance of WT1 expression in tumor cells is associated with a good prognosis in malignant glioma patients treated with WT1 peptide vaccine immunotherapy

doi: 10.1007/s00262-021-02954-z

Figure Lengend Snippet: Summary of IHC staining results of both pre- and post- WT1 vaccination specimens

Article Snippet: The sections were then incubated overnight at 4 °C with anti-human WT1 mouse monoclonal antibody 6F-H2 (DAKO, Carpinteria, CA, USA; diluted 1:50), anti-human Transforming growth factor-β (TGF-β) rabbit polyclonal antibody (Abcam, Cambridge, MA, USA; diluted 1:200), anti–human Ki-67 mouse monoclonal antibody MIB-1 (DAKO; diluted 1:40), anti-human HLA class I-ABC mouse monoclonal antibody EMR8-5 (Hokudo, Sapporo, Japan; diluted 1:100), anti-human programmed death-ligand 1 (PD-L1) rabbit monoclonal antibody ab205921 clone 28–8, (Abcam; diluted 1:100), anti-human CD3 mouse monoclonal antibody F7.2.38 (DAKO; diluted 1:5), anti–human CD4 mouse monoclonal antibody 4B12 (DAKO; diluted 1:80), anti–human CD8 mouse monoclonal antibody C8/14B (Nichirei Biosciences, Tokyo, Japan; undiluted), anti-human CD79α mouse monoclonal antibody JCB117 (DAKO; diluted 1:200), anti-human PD-1 mouse monoclonal antibody clone NAT105 (Abcam; diluted 1:100), anti-human Foxp3 mouse monoclonal antibody 236A/E7 (Abcam; diluted 1:100), or anti IDH1­R132H mouse monoclonal Antibody (FUJIFILM Wako Chemicals, Osaka, Japan; diluted 1:100).

Techniques: Immunohistochemistry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Maintenance of WT1 expression in tumor cells is associated with a good prognosis in malignant glioma patients treated with WT1 peptide vaccine immunotherapy

doi: 10.1007/s00262-021-02954-z

Figure Lengend Snippet: Changes in the expression of WT1, HLA class I, TGF-β, PD-L1, and TIL count pre- and post- WT1 vaccination. The median WT1 score, TGF-β score, HLA class I staining index, and PD-L1 score were calculated. The median number of CD4+, CD8+, PD-1+, and Foxp3+ TILs per square millimeter were counted. These data were compared between tumor samples resected before and after WT1 vaccination. In this figure, the distribution of WT1 score, HLA class I staining index, TGF-β score, PD-L1 score, and TIL count are represented by the box plot, and the limits of the whiskers indicate the extremes and those of the box the 25 and 75% values. WT1 score, HLA class I staining index, and CD4+ TILs were significantly reduced in tumor specimens post- WT1 vaccination. In contrast, the TGF-β score was increased, while the PD-L1 score, CD8+ TILs, PD-1+ TILs, and Foxp3+ TILs were not significantly different (*P < 0.05)

Article Snippet: The sections were then incubated overnight at 4 °C with anti-human WT1 mouse monoclonal antibody 6F-H2 (DAKO, Carpinteria, CA, USA; diluted 1:50), anti-human Transforming growth factor-β (TGF-β) rabbit polyclonal antibody (Abcam, Cambridge, MA, USA; diluted 1:200), anti–human Ki-67 mouse monoclonal antibody MIB-1 (DAKO; diluted 1:40), anti-human HLA class I-ABC mouse monoclonal antibody EMR8-5 (Hokudo, Sapporo, Japan; diluted 1:100), anti-human programmed death-ligand 1 (PD-L1) rabbit monoclonal antibody ab205921 clone 28–8, (Abcam; diluted 1:100), anti-human CD3 mouse monoclonal antibody F7.2.38 (DAKO; diluted 1:5), anti–human CD4 mouse monoclonal antibody 4B12 (DAKO; diluted 1:80), anti–human CD8 mouse monoclonal antibody C8/14B (Nichirei Biosciences, Tokyo, Japan; undiluted), anti-human CD79α mouse monoclonal antibody JCB117 (DAKO; diluted 1:200), anti-human PD-1 mouse monoclonal antibody clone NAT105 (Abcam; diluted 1:100), anti-human Foxp3 mouse monoclonal antibody 236A/E7 (Abcam; diluted 1:100), or anti IDH1­R132H mouse monoclonal Antibody (FUJIFILM Wako Chemicals, Osaka, Japan; diluted 1:100).

Techniques: Expressing, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Maintenance of WT1 expression in tumor cells is associated with a good prognosis in malignant glioma patients treated with WT1 peptide vaccine immunotherapy

doi: 10.1007/s00262-021-02954-z

Figure Lengend Snippet: Univariate analysis using a Cox proportional hazards model for overall survival and progression free survival from the start of WT1 vaccination

Article Snippet: The sections were then incubated overnight at 4 °C with anti-human WT1 mouse monoclonal antibody 6F-H2 (DAKO, Carpinteria, CA, USA; diluted 1:50), anti-human Transforming growth factor-β (TGF-β) rabbit polyclonal antibody (Abcam, Cambridge, MA, USA; diluted 1:200), anti–human Ki-67 mouse monoclonal antibody MIB-1 (DAKO; diluted 1:40), anti-human HLA class I-ABC mouse monoclonal antibody EMR8-5 (Hokudo, Sapporo, Japan; diluted 1:100), anti-human programmed death-ligand 1 (PD-L1) rabbit monoclonal antibody ab205921 clone 28–8, (Abcam; diluted 1:100), anti-human CD3 mouse monoclonal antibody F7.2.38 (DAKO; diluted 1:5), anti–human CD4 mouse monoclonal antibody 4B12 (DAKO; diluted 1:80), anti–human CD8 mouse monoclonal antibody C8/14B (Nichirei Biosciences, Tokyo, Japan; undiluted), anti-human CD79α mouse monoclonal antibody JCB117 (DAKO; diluted 1:200), anti-human PD-1 mouse monoclonal antibody clone NAT105 (Abcam; diluted 1:100), anti-human Foxp3 mouse monoclonal antibody 236A/E7 (Abcam; diluted 1:100), or anti IDH1­R132H mouse monoclonal Antibody (FUJIFILM Wako Chemicals, Osaka, Japan; diluted 1:100).

Techniques: Mutagenesis

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Maintenance of WT1 expression in tumor cells is associated with a good prognosis in malignant glioma patients treated with WT1 peptide vaccine immunotherapy

doi: 10.1007/s00262-021-02954-z

Figure Lengend Snippet: Association between the prognostic markers and overall survival from the start of the WT1 vaccination (OS-WT1) is shown using Kaplan–Meier survival curves. The log-rank test was used to compare the differences

Article Snippet: The sections were then incubated overnight at 4 °C with anti-human WT1 mouse monoclonal antibody 6F-H2 (DAKO, Carpinteria, CA, USA; diluted 1:50), anti-human Transforming growth factor-β (TGF-β) rabbit polyclonal antibody (Abcam, Cambridge, MA, USA; diluted 1:200), anti–human Ki-67 mouse monoclonal antibody MIB-1 (DAKO; diluted 1:40), anti-human HLA class I-ABC mouse monoclonal antibody EMR8-5 (Hokudo, Sapporo, Japan; diluted 1:100), anti-human programmed death-ligand 1 (PD-L1) rabbit monoclonal antibody ab205921 clone 28–8, (Abcam; diluted 1:100), anti-human CD3 mouse monoclonal antibody F7.2.38 (DAKO; diluted 1:5), anti–human CD4 mouse monoclonal antibody 4B12 (DAKO; diluted 1:80), anti–human CD8 mouse monoclonal antibody C8/14B (Nichirei Biosciences, Tokyo, Japan; undiluted), anti-human CD79α mouse monoclonal antibody JCB117 (DAKO; diluted 1:200), anti-human PD-1 mouse monoclonal antibody clone NAT105 (Abcam; diluted 1:100), anti-human Foxp3 mouse monoclonal antibody 236A/E7 (Abcam; diluted 1:100), or anti IDH1­R132H mouse monoclonal Antibody (FUJIFILM Wako Chemicals, Osaka, Japan; diluted 1:100).

Techniques:

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Differential Expression of Wilms’ Tumor Protein in Diffuse Intrinsic Pontine Glioma

doi: 10.1093/jnen/nlz021

Figure Lengend Snippet: Summary of Pediatric CNS Tumor Patient Samples Used in This Study

Article Snippet: Mouse monoclonal anti-WT1 (6F-H2, Abcam, Cambridge, MA), rabbit polyclonal anti-histone H3K27M and rabbit monoclonal anti-tri-methyl-histone H3 (H3K27me3) (C36B11, Cell Signaling Technology, Danvers, MA) were diluted in Bond primary antibody diluent (#AR9352 Leica Biosystems, Buffalo Grove, IL) to concentrations of 1:15, 1:200, and 1:400, respectively.

Techniques:

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Differential Expression of Wilms’ Tumor Protein in Diffuse Intrinsic Pontine Glioma

doi: 10.1093/jnen/nlz021

Figure Lengend Snippet: WT1 expression is associated with pediatric CNS cancers. (A) Analysis of publicly available gene expression profiles of a larger cohort of pediatric GBMs. This analysis showed that WT1 is highly expressed in pediatric GBMs (n = 74) compared with pediatric healthy brain specimens (n = 22) (2.9-fold, p = 0.023). (B) qRT-PCR using cDNA from DIPG specimens validated WT1 overexpression in tumor (n = 5) versus normal (n = 5) within each patient (4.1-fold). (C) WT1 overexpression in tumor tissues was validated by Western blot assays using DIPG tumor specimens (n = 9) and adjacent normal specimens (n = 9). Quantification of the Western blot assays of DIPG tumor versus normal showed significantly higher expression of WT1 in the tumors (7.4-fold, p < 0.0001).

Article Snippet: Mouse monoclonal anti-WT1 (6F-H2, Abcam, Cambridge, MA), rabbit polyclonal anti-histone H3K27M and rabbit monoclonal anti-tri-methyl-histone H3 (H3K27me3) (C36B11, Cell Signaling Technology, Danvers, MA) were diluted in Bond primary antibody diluent (#AR9352 Leica Biosystems, Buffalo Grove, IL) to concentrations of 1:15, 1:200, and 1:400, respectively.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Western Blot

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Differential Expression of Wilms’ Tumor Protein in Diffuse Intrinsic Pontine Glioma

doi: 10.1093/jnen/nlz021

Figure Lengend Snippet: Demographic and clinical distribution of patients used for the study. Formalin-fixed paraffin-embedded (FFPE) specimens from a large cohort of pediatric CNS tumors (n = 41) were used in this study to examine the expression of WT1 in the tumors. The tumor grade of each specimen is represented by the orange shades. The driver and partner mutations were established using droplet PCR or WES. The specimens were checked for H3 mutation status by IHC only (n = 20; dark green boxes) or by either droplet PCR or WES (n = 16).

Article Snippet: Mouse monoclonal anti-WT1 (6F-H2, Abcam, Cambridge, MA), rabbit polyclonal anti-histone H3K27M and rabbit monoclonal anti-tri-methyl-histone H3 (H3K27me3) (C36B11, Cell Signaling Technology, Danvers, MA) were diluted in Bond primary antibody diluent (#AR9352 Leica Biosystems, Buffalo Grove, IL) to concentrations of 1:15, 1:200, and 1:400, respectively.

Techniques: Formalin-fixed Paraffin-Embedded, Expressing, Mutagenesis

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Differential Expression of Wilms’ Tumor Protein in Diffuse Intrinsic Pontine Glioma

doi: 10.1093/jnen/nlz021

Figure Lengend Snippet: Immunohistochemistry of a large cohort of DIPGs indicated high expression of WT1 in H3.3 subtype compared with H3.1 subtype DIPGs. (A) IHC of a large cohort of DIPGs and pediatric midline gliomas showed WT1 overexpression in tumor compared with adjacent healthy brain tissue was valid. Scale bar = 50 µm. (B) Each tumor specimen was reviewed by a blinded neuropathologist and given an immunoreactivity score based on the number of WT1-positive cells. The pie chart shows the WT1 scores distribution of the specimens. (C) Examination of WT1 immunoreactivity scores revealed that WT1 scores of the specimens are associated with histone H3 mutation status. The distribution of WT1 immunoreactivity score was significantly different between H3.3 and H3.1 subtypes (Fisher exact test, p = 0.017). (D) qRT-PCR using cDNA from H3.3K27M DIPG tumor tissue (n = 3) and H3.1K27M DIPG tumor tissue (n = 2) showed higher expression of WT1 in H3.3K27M tumor specimens compared with H3.1K27M tumor specimens (6.4-fold).

Article Snippet: Mouse monoclonal anti-WT1 (6F-H2, Abcam, Cambridge, MA), rabbit polyclonal anti-histone H3K27M and rabbit monoclonal anti-tri-methyl-histone H3 (H3K27me3) (C36B11, Cell Signaling Technology, Danvers, MA) were diluted in Bond primary antibody diluent (#AR9352 Leica Biosystems, Buffalo Grove, IL) to concentrations of 1:15, 1:200, and 1:400, respectively.

Techniques: Immunohistochemistry, Expressing, Over Expression, Mutagenesis, Quantitative RT-PCR

Journal: Journal of Neuropathology and Experimental Neurology

Article Title: Differential Expression of Wilms’ Tumor Protein in Diffuse Intrinsic Pontine Glioma

doi: 10.1093/jnen/nlz021

Figure Lengend Snippet: Validation of WT1 differential expression in human DIPG primary cells. (A) A Western blot assay of WT1 using human DIPG primary cells (n = 6) and healthy brain tissue (HBT) lysates. (B) Quantification of the Western blot assays showed significantly higher WT1 in human DIPG primary cells compared with healthy brain tissue (3.63-fold, p = 0.047). (C) Human DIPG primary cells harboring H3.3K27M showed higher WT1 compared with human DIPG primary cells harboring H3.1K27M (2.83-fold) and histone H3 wildtype (2.86-fold). (D) Immunofluorescence staining of human DIPG primary cells (n = 2) showed WT1 expression in the tumor cells and localization of WT1, mainly in the cytoplasm of the tumor cells. The specificity of the antibody was validated by staining K562 cells, which showed WT1 localization in the nucleus. Scale bar = 30 µm.

Article Snippet: Mouse monoclonal anti-WT1 (6F-H2, Abcam, Cambridge, MA), rabbit polyclonal anti-histone H3K27M and rabbit monoclonal anti-tri-methyl-histone H3 (H3K27me3) (C36B11, Cell Signaling Technology, Danvers, MA) were diluted in Bond primary antibody diluent (#AR9352 Leica Biosystems, Buffalo Grove, IL) to concentrations of 1:15, 1:200, and 1:400, respectively.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining