rabbit anti human phospho her2 erbb2 tyr1221 1222  (Cell Signaling Technology Inc)

Journal: Cancers

Article Title: Characterization of HER2-Positive Murine Breast Cancer Models for Investigating HER2-Targeted Therapy and Immunotherapy

doi: 10.3390/cancers18060997

Figure Lengend Snippet: Validation of HER2 expression in the newly established HER2-positive mouse tumors. ( A ) Western blotting analysis of HER2 and phosphor-HER2 in transduced 4T1 and EO771 cell lines. BT474 included as HER2+ control. ( B ) Representative immunofluorescence staining of HER2 expression in transduced EO771 cells. ( C ) Tumor volume curve of 4T1 ( top ) and EO771 ( bottom ) HER2-OE lines (n = 4–5 per group). Data is mean ± SD. ( D ) Representative immunohistochemistry staining of HER2 expression on tumor slices. The uncropped blots are included in .

Article Snippet: Briefly, for IF, cells grown in 96-well plates were fixed with 10% formalin for 30 min at room temperature and blocked with 1% BSA in PBS for 1 h. Cells were incubated overnight at 4 °C with primary rabbit anti-human HER2 antibody (Cell Signaling Technology, 2242S, 1:100 dilution), followed by incubation with Cy5-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, 711-175-152, 1:200) for 1 h at room temperature in the dark.

Techniques: Biomarker Discovery, Expressing, Western Blot, Control, Immunofluorescence, Staining, Immunohistochemistry

Journal: Cancers

Article Title: Characterization of HER2-Positive Murine Breast Cancer Models for Investigating HER2-Targeted Therapy and Immunotherapy

doi: 10.3390/cancers18060997

Figure Lengend Snippet: Trastuzumab alone was not able to reduce primary tumor size but decreased tumor brain metastasis in 4T1-HER2 models. ( A ) Tumor volume curve of 4T1-GFP, -HER2 WT , and -HER2 YVMA tumors treated with either saline or trastuzumab (15 mg/kg, IV). Filled shapes represent saline-treated groups; open shapes represent trastuzumab-treated groups. No statistically significant difference was observed between groups (repeated measures ANOVA p = 0.56). ( B ) Plot of average radiance from GFP fluorescence in the brains of 4T1 tumor-bearing mice. Symbols represent individual mice. ( C ) Fluorescence images of the brains of 4T1 tumor-bearing mice. TRA, trastuzumab. Data is shown as mean ± SD. Two-way ANOVA with Tukey’s HSD test was used in ( B ). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Briefly, for IF, cells grown in 96-well plates were fixed with 10% formalin for 30 min at room temperature and blocked with 1% BSA in PBS for 1 h. Cells were incubated overnight at 4 °C with primary rabbit anti-human HER2 antibody (Cell Signaling Technology, 2242S, 1:100 dilution), followed by incubation with Cy5-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, 711-175-152, 1:200) for 1 h at room temperature in the dark.

Techniques: Saline, Fluorescence

Journal: Cancers

Article Title: Characterization of HER2-Positive Murine Breast Cancer Models for Investigating HER2-Targeted Therapy and Immunotherapy

doi: 10.3390/cancers18060997

Figure Lengend Snippet: HER2-targeted therapy synergizes with immunotherapy in HER2-overexpressing (OE) tumors. ( A ) Tumor volume ( left ) and survival ( right ) curves for EO771-HER2 YVMA and 4T1-HER2 YVMA models treated with saline, anti-HER2 agents (TRA + TUC), ICB (αPD-1 and αCTLA-4), or combination therapy (anti-HER2 + ICB) as indicated by arrows. ( B , C ) Flow cytometry quantification of CD4+ ( B ) and CD8+ ( C ) T cells in EO771-HER2 YVMA tumors on day 5. Mantel–Cox logrank test was used for survival analysis in ( A ). Two-way ANOVA with Bonferroni correction for multiple comparisons was used for ( B , C ). All data are shown as mean ± SD; * p < 0.05; ** p < 0.01.

Article Snippet: Briefly, for IF, cells grown in 96-well plates were fixed with 10% formalin for 30 min at room temperature and blocked with 1% BSA in PBS for 1 h. Cells were incubated overnight at 4 °C with primary rabbit anti-human HER2 antibody (Cell Signaling Technology, 2242S, 1:100 dilution), followed by incubation with Cy5-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, 711-175-152, 1:200) for 1 h at room temperature in the dark.

Techniques: Saline, Flow Cytometry

Journal: Cancers

Article Title: Characterization of HER2-Positive Murine Breast Cancer Models for Investigating HER2-Targeted Therapy and Immunotherapy

doi: 10.3390/cancers18060997

Figure Lengend Snippet: HER2-overexpressing models show differential responses to T-DM1 and T-Dxd. Tumor volume curves for BT474 (human HER2+) ( A ), HER2− MDA-MB-231 (human TNBC) ( B ), 4T1-HER2 WT ( C ), and EO771-HER2 WT ( D ) treated with saline (black line), T-DM1 (blue line), or T-Dxd (red line). n = 3 per group. * p < 0.05.

Article Snippet: Briefly, for IF, cells grown in 96-well plates were fixed with 10% formalin for 30 min at room temperature and blocked with 1% BSA in PBS for 1 h. Cells were incubated overnight at 4 °C with primary rabbit anti-human HER2 antibody (Cell Signaling Technology, 2242S, 1:100 dilution), followed by incubation with Cy5-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, 711-175-152, 1:200) for 1 h at room temperature in the dark.

Techniques: Saline

Journal: NPJ Precision Oncology

Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

doi: 10.1038/s41698-026-01365-6

Figure Lengend Snippet: A TTNT with T-DXd by HER2 status of the disease; B TTNT with T-DXd by clinical subtype of the disease; C TTNT with T-DXd depending on changes in HER2 status (low vs. 0) between the primary tumor and last metastatic biopsy; D TTNT with T-DXd depending on changes in HER2 status (low vs. 0) between the first and most recent metastatic biopsy; E OS with T-DXd by HER2 status of the disease; F OS with T-DXd by clinical subtype of the disease. T-DXd, trastuzumab deruxtecan; TTNT, time to next treatment; HER2, human epidermal growth factor receptor 2; OS, overall survival; HR, hormone receptor; TNBC, triple-negative breast cancer.

Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

Techniques:

Journal: NPJ Precision Oncology

Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

doi: 10.1038/s41698-026-01365-6

Figure Lengend Snippet: Samples are ordered by HER2 IHC subtype and TTNT from the start of T-DXd treatment. Clinical parameters displayed include HER2 IHC subtype (HER2-zero, HER2-low, HER2-positive), latest HER2 IHC prior to T-DXd, number of prior lines of chemotherapy, and receipt of sacituzumab govitecan before T-DXd. An asterisk on the TTNT bar indicates treatment is still ongoing. Bar plots show scores for each assay, and these values are also represented with color-coded squares, along with their corresponding quartiles for the HS-HER2 and RPPA HER2 assays. For HER2DX ERBB2, score groups are displayed based on the recommended cutoffs: Low (1–32), Med (33–50), and High (51–99) . Some cases are depicted as HER2-positive given prior HER2-positivity, despite the closest biopsy to T-DXd administration being formally HER2-negative (as suggested by the IHC score). HS-HER2, High Sensitivity-human epidermal growth factor receptor 2; RPPA, Reverse Phase Protein Array; T-DXd, trastuzumab deruxtecan; TTNT, time to next treatment. *did not experience TTNT event.

Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

Techniques: Protein Array

Journal: NPJ Precision Oncology

Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

doi: 10.1038/s41698-026-01365-6

Figure Lengend Snippet: A Workflow for the quantification of HS-HER2; B TTNT with T-DXd according to HS-HER2 quartiles; C OS with T-DXd according to HS-HER2 quartiles; D TTNT with T-DXd according to HS-HER2 median (HER2-positive disease only); E TTNT with T-DXd in the HS-HER2 cohort according to the traditional HER2 IHC classification of HER2-positive, HER2-low-HER2-0 in the HS-HER2 population. T-DXd trastuzumab deruxtecan, TTNT time to next treatment, HS-HER2 High Sensitivity-HER2, IHC immunohistochemistry, ROI region of interest, OS overall survival.

Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

Techniques: Immunohistochemistry

Journal: NPJ Precision Oncology

Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

doi: 10.1038/s41698-026-01365-6

Figure Lengend Snippet: A Workflow for the CLIA-based RPPA analysis; B TTNT with T-DXd according to RPPA quantified total HER2 protein quartiles; C . OS with T-DXd according to RPPA HER2 protein expression quartiles; D TTNT with T-DXd according to RPPA measured HER2 activation (phosphoHER2 Y1248) quartiles; E OS with T-DXd according to RPPA measured HER2 activation (phosphoHER2 Y1248) quartiles; T-DXd trastuzumab deruxtecan, CLIA-RPPA Clinical Laboratory Improvement Amendments- Reverse Phase Protein Array, TTNT time to next treatment, OS overall survival.

Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

Techniques: Expressing, Activation Assay, Protein Array

Journal: NPJ Precision Oncology

Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

doi: 10.1038/s41698-026-01365-6

Figure Lengend Snippet: A TTNT with T-DXd according to RPPA measured TOPO1 protein expression median (HER2-negative only); B OS with T-DXd according to RPPA measured TOPO1 protein expression median (HER2-negative only); C TTNT with T-DXd according to RPPA Trop2 quartiles; D TTNT with T-DXd according to RPPA measured EGFR protein expression quartiles; E TTNT with T-DXd according to RPPA measured phosphoHER3 protein expression quartiles; F TTNT with T-DXd according to the traditional HER2 IHC classification of HER2-positive, HER2-low-HER2-0 in the RPPA population. T-DXd trastuzumab deruxtecan, TTNT time to next treatment, RPPA Reverse Phase Protein Array, OS overall survival.

Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

Techniques: Expressing, Protein Array

Journal: NPJ Precision Oncology

Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

doi: 10.1038/s41698-026-01365-6

Figure Lengend Snippet: A Description of HER2DX gene expression modules; association between the HER2 amplicon module with TTNT ( B ) and OS in all patients ( C ); association between the HER2 amplicon module with TTNT ( D ) and OS in HER2-positive MBC ( E ); F association between TTNT and the luminal module in patients with HER2-negative disease; G TTNT with T-DXd according to the traditional HER2 IHC classification of HER2-positive, HER2-low-HER2-0 in the HER2DX population. TTNT time to next treatment, OS overall survival, MBC metastatic breast cancer, T-DXd trastuzumab deruxtecan, IHC immunohistochemistry.

Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

Techniques: Gene Expression, Amplification, Immunohistochemistry

Journal: NPJ Precision Oncology

Article Title: Quantitative HER2 tissue and plasma profiling predicts the activity of trastuzumab deruxtecan for breast cancer

doi: 10.1038/s41698-026-01365-6

Figure Lengend Snippet: A Description of DNADX workflow; B Outcomes with T-DXd for metastatic breast cancer according to DNADX-detected tumor fraction; C Outcomes with T-DXd for metastatic breast cancer according to DNADX subtype; D Outcomes with T-DXd for metastatic breast cancer according to the DNADX HER2 signature; E Outcomes with T-DXd for metastatic breast cancer according to HER2 IHC status in the cohort of patients with detectable tumor fraction. T-DXd trastuzumab deruxtecan, TTNT, time to next treatment, OS overall survival.

Article Snippet: The Leica BOND Rx autostainer protocol is as follows: deparaffinization with BOND dewax solution (AR9222), antigen retrieval with BOND HIER epitope retrieval solution 2 (AR9640) at 97 ° C for 20 min, blocking with ReadyProbes Endogenous HRP & AP blocking solution ( R37629 , Invitrogen) for 10 min and with BSA for 30 min, 1 h incubation with primary rabbit monoclonal HER2 antibody (clone 29D8, #2165, IgG, Cell Signaling) at optimal concentration of 1ug/ml mixed together with 1:100 concentration of pan-CK (Clones AE1/AE3, REF#M3515, Dako), amplification with Rabbit Envision+ System–HRP labeled polymer anti-Rabbit (K400311-2, Dako) mixed together with 1:100 dilution of a green-fluorescent Alexa Fluor 546 Goat-anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (REF#A11003) for 1 h, staining with 1:50 dilution of a red-fluorescent Tyramide Signal Amplification (TSA) Cyanin 5 (SAT705A001EA, Akoya Biosciences) for 10 min and nuclear staining with 1:500 dilution of a blue-fluorescent 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.

Techniques: