Antibodies used in this study
. | . | . | . | . | Specificity testing . | . | . | . | ||
|---|---|---|---|---|---|---|---|---|---|---|
| Antibody specificity . | Supplier and antibody . | Antigen retrieval . | Incubation time, h* . | Dilution . | Blocking . | Western blot . | Staining . | Remarks . | ||
| PDGFRA | Santa Cruz, C-20 | 1-min PC | 3-18 | 1:50 | Yes | Yes | Fibroblasts in several tissues | - | ||
| PDGFRA | Dianova, DLN1357 | 15-min MWO | 1 | 1:100 | ND | Yes | Fibroblasts in several tissues | Used for confirmation; same peptide used for immunization as for Santa Cruz antibody | ||
| DDR2 | Santa Cruz, H-108 | 30-min MWO | 18 | 1:50 | NA | Yes | Fibroblasts in B-NHL | - | ||
| DDR2 | Genex, GEA4023, Bioscience | 15-min MWO | 18 | 1:250 | Yes | No | ND | Used for confirmation | ||
| RON | Santa Cruz, C-20 | 1-min PC | 2-18 | 1:50 | Yes | Yes | Occasional staining of macrophages and dendritic cells in lymphatic tissues | - | ||
| TRKA† | Santa Cruz, C-14 | 1-min PC | 2-18 | 1:100 | Yes | Yes | Brain, granulocytes in lymphatic tissues | - | ||
| TRKA | Oncogene, PC31 | 30-min MWO | 2-18 | 1:50 | Yes | Yes | Brain, granulocytes in lymphatic tissues | Used for confirmation; same peptide used for immunization as for Santa Cruz antibody | ||
| EPHB1 | Santa Cruz, Q-20 | 1-min PC | 2-18 | 1:50 | Yes | No bands with positive control | Hippocampal neurons, occasionally histiocytes in B-NHL | - | ||
| EPHB1 | Santa Cruz, M19 | NA | 18 | NA | NA | Yes | ND | Used for Western Blot | ||
| TRKB | Santa Cruz, 794 | 15-min MWO | 2-18 | 1:50 | Yes | No bands with positive control | Brain, granulocytes in lymphatic tissues | - | ||
| PDGFA | Santa Cruz, N30 | 15-min MWO | 3 | 1:50 | Yes | Yes | Kidney tubuli | - | ||
| PDGFA | Santa Cruz, E10 | No | 18 | 1:30 | NA | Yes | Kidney tubuli | Used for confirmation | ||
| Collagen 1 | Acris, AF5610 | 15-min MWO | 1 | 1:50 | NA | ND | Staining of fibroblasts and extracellular matrix | - | ||
| Collagen 1 | Abcam, Ab-6308 | 15-min MWO | 1 | 1:500 | NA | ND | Staining of fibroblasts and extracellular matrix | Used for confirmation | ||
| NGF | Abcam, Ab-6198 | 15-min MWO | 3 | 1:200 | NA | ND | Staining of several neurons in brain and occasional granulocytes in lymphatic tissues | - | ||
| Ephrin B1 | Santa Cruz, A20 | 15-min MWO | 3 | 1:50 | Yes | ND | Staining of corpus luteum | - | ||
| Ephrin B1 | Zymed, ZMD.41 | Pepsin | 3 | 1:50 | NA | ND | Staining of corpus luteum | Used for confirmation | ||
| Pan-p-Y | Upstate, 4G10 | 15-min MWO | 18 | 1:1000 | NA | NA | ALK+ anaplastic large-cell lymphomas, KIT+ gastrointestinal stromal tumors | Preincubation of sections with T-cell phosphatase strongly decreased or abolished staining completely | ||
| Pan-p-Y | Sigma, PY20 | 15-min MWO | 3 | 1:100 | NA | NA | ALK+ anaplastic large-cell lymphomas | - | ||
| Pan-p-Y | Sigma, PY99 | 15-min MWO | 3 | 1:50 | NA | NA | ALK+ anaplastic large-cell lymphomas | - | ||
| p-Y-720-PDGFRA | Santa Cruz, 12910-R | 15-min MWO | 3 | 1:50 | Yes | Yes | Specificity tested against ALK+ ALCLs and TRK+ but PDGFRA- Hodgkin lymphoma patients | Preincubation of sections with T-cell phosphatase strongly decreased or abolished staining completely | ||
| p-Y-680/681-TRKA/B | Santa Cruz, 7996-R | 15-min MWO | 3 | 1:50 | Yes | No | Specificity tested against ALK+ ALCLs and PDGFRA+ but TRKA/B- Hodgkin lymphoma patients | Preincubation of sections with T-cell phosphatase strongly decreased or abolished staining completely | ||
. | . | . | . | . | Specificity testing . | . | . | . | ||
|---|---|---|---|---|---|---|---|---|---|---|
| Antibody specificity . | Supplier and antibody . | Antigen retrieval . | Incubation time, h* . | Dilution . | Blocking . | Western blot . | Staining . | Remarks . | ||
| PDGFRA | Santa Cruz, C-20 | 1-min PC | 3-18 | 1:50 | Yes | Yes | Fibroblasts in several tissues | - | ||
| PDGFRA | Dianova, DLN1357 | 15-min MWO | 1 | 1:100 | ND | Yes | Fibroblasts in several tissues | Used for confirmation; same peptide used for immunization as for Santa Cruz antibody | ||
| DDR2 | Santa Cruz, H-108 | 30-min MWO | 18 | 1:50 | NA | Yes | Fibroblasts in B-NHL | - | ||
| DDR2 | Genex, GEA4023, Bioscience | 15-min MWO | 18 | 1:250 | Yes | No | ND | Used for confirmation | ||
| RON | Santa Cruz, C-20 | 1-min PC | 2-18 | 1:50 | Yes | Yes | Occasional staining of macrophages and dendritic cells in lymphatic tissues | - | ||
| TRKA† | Santa Cruz, C-14 | 1-min PC | 2-18 | 1:100 | Yes | Yes | Brain, granulocytes in lymphatic tissues | - | ||
| TRKA | Oncogene, PC31 | 30-min MWO | 2-18 | 1:50 | Yes | Yes | Brain, granulocytes in lymphatic tissues | Used for confirmation; same peptide used for immunization as for Santa Cruz antibody | ||
| EPHB1 | Santa Cruz, Q-20 | 1-min PC | 2-18 | 1:50 | Yes | No bands with positive control | Hippocampal neurons, occasionally histiocytes in B-NHL | - | ||
| EPHB1 | Santa Cruz, M19 | NA | 18 | NA | NA | Yes | ND | Used for Western Blot | ||
| TRKB | Santa Cruz, 794 | 15-min MWO | 2-18 | 1:50 | Yes | No bands with positive control | Brain, granulocytes in lymphatic tissues | - | ||
| PDGFA | Santa Cruz, N30 | 15-min MWO | 3 | 1:50 | Yes | Yes | Kidney tubuli | - | ||
| PDGFA | Santa Cruz, E10 | No | 18 | 1:30 | NA | Yes | Kidney tubuli | Used for confirmation | ||
| Collagen 1 | Acris, AF5610 | 15-min MWO | 1 | 1:50 | NA | ND | Staining of fibroblasts and extracellular matrix | - | ||
| Collagen 1 | Abcam, Ab-6308 | 15-min MWO | 1 | 1:500 | NA | ND | Staining of fibroblasts and extracellular matrix | Used for confirmation | ||
| NGF | Abcam, Ab-6198 | 15-min MWO | 3 | 1:200 | NA | ND | Staining of several neurons in brain and occasional granulocytes in lymphatic tissues | - | ||
| Ephrin B1 | Santa Cruz, A20 | 15-min MWO | 3 | 1:50 | Yes | ND | Staining of corpus luteum | - | ||
| Ephrin B1 | Zymed, ZMD.41 | Pepsin | 3 | 1:50 | NA | ND | Staining of corpus luteum | Used for confirmation | ||
| Pan-p-Y | Upstate, 4G10 | 15-min MWO | 18 | 1:1000 | NA | NA | ALK+ anaplastic large-cell lymphomas, KIT+ gastrointestinal stromal tumors | Preincubation of sections with T-cell phosphatase strongly decreased or abolished staining completely | ||
| Pan-p-Y | Sigma, PY20 | 15-min MWO | 3 | 1:100 | NA | NA | ALK+ anaplastic large-cell lymphomas | - | ||
| Pan-p-Y | Sigma, PY99 | 15-min MWO | 3 | 1:50 | NA | NA | ALK+ anaplastic large-cell lymphomas | - | ||
| p-Y-720-PDGFRA | Santa Cruz, 12910-R | 15-min MWO | 3 | 1:50 | Yes | Yes | Specificity tested against ALK+ ALCLs and TRK+ but PDGFRA- Hodgkin lymphoma patients | Preincubation of sections with T-cell phosphatase strongly decreased or abolished staining completely | ||
| p-Y-680/681-TRKA/B | Santa Cruz, 7996-R | 15-min MWO | 3 | 1:50 | Yes | No | Specificity tested against ALK+ ALCLs and PDGFRA+ but TRKA/B- Hodgkin lymphoma patients | Preincubation of sections with T-cell phosphatase strongly decreased or abolished staining completely | ||
Specificity was tested by blocking with peptide used for immunization, by Western blot analysis with HRS cell lines and positive controls from supplier, and by staining in a positive control.
NA indicates not applicable; ND, not done; PC, pressure cooking for 1 minute at 121°C in 1 mM EDTA (ethylenediaminetetraacetic acid) pH 8.0; MWO, 15 or 30 minutes in a microwave oven at maximum power in 600 mL of 10 mM Na citrate pH 6.0.
Suppliers were as follows: Santa Cruz Biotechnology, Santa Cruz, CA; Dianova, Hamburg, Germany; Genex Bioscience, Hayward, CA; Oncogene, Boston, MA; Acris, Hiddenhausen, Germany; Abcam, Cambridge, United Kingdom; Zymed, San Francisco, CA; Upstate, Waltham, MA; Sigma, Munich, Germany.
All incubations were performed at room temperature
As stated by the supplier, this antibody should also react with TRKB and TRKC. However, in WB, the KMH2 cell line, which showed a strong signal for TRKB expression in the microarray analysis, was negative. Therefore, we regarded this anti-TRK antibody as TRKA specific