51-3900

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of ALCL tissue using ALK Polyclonal Antibody, Rabbit, (PAD: ZAL4). Image courtesy of Dr. G. Inghirami, NYU.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of ALK showing staining in the cytoplasm of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a ALK polyclonal antibody (Product # 51-3900) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of ALK showing staining in the cytoplasm of paraffin-embedded human skeletal muscle tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ALK Polyclonal Antibody (Product # 51-3900) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 Enhanced DNMT1 expression is detected in human ALCL cell lines and patient samples. A. DNMT1 is overexpressed in human ALCL cell lines compared to normal peripheral blood mononuclear cells (PBMCs). Proteins were extracted from PBMCs, ALK- ALCL cell line MAC-2A and ALK+ ALCL cell lines KARPAS-299 and SR-786 and analysed by Western Blot as described in materials and methods. B. DNMT1 is expressed in human ALK+ ALCL tumor cells, but not in healthy lymph nodes. Immunohistochemical staining against DNMT1 was performed on a human ALCL tissue array containing 30 ALK+ and 5 ALK- ALCL patient samples and 7 normal lymph nodes as controls. One representative lymph node, one representative ALK+ and one ALK- ALCL sample are shown. CD30 is used as diagnostic marker for ALCL. Note the typical abnormal anaplastic large cell morphology of CD30 positive cells compared to healthy lymphocytes.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 ALK-F1174L impairs differentiation of MONC-1-derived tumors A. Whole cell extract of MONC-1 parental cells and MONC-1-ALK-F1174L transduced cells were analyzed by immunoblotting for the presence of human ALK. beta-actin was used as loading control. B. Tumor take (number of tumor-bearing mice /total nude mice) and growth (mean tumor volumes +- SEM) of MONC-1 and MONC-1-ALK-F cells orthotopically implanted and measured by echography (unpaired t test with Welch's correction, ***=p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 ALK-wt-, ALK-F1174L-, and ALK-R1275Q-expressing JoMa1 cells are tumorigenic in subcutaneous in vivo model A. Analyses of ALK mRNA and protein expressions in parental JoMa1 cells and in JoMa1 cells transduced with the empty vector (Migr) or vector encoding for ALF-wt, ALK-F1174L or ALK-R1275Q. Murine GAPDH and beta-actin were used as control for RT-PCR (top) or immunoblotting (bottom), respectively. B. Expression levels of various NCSC and differentiation markers analyzed by RT-PCR in total RNA from JoMa1 parental and transduced cells. C. Immunoblotting analysis of ALK phosphorylation in transduced JoMa1 cells. beta-actin was used as loading control. D. Tumor take (number of tumors/total implanted mouse flanks) and growth (mean tumor volumes +- SEM) of JoMa1 parental and transduced cells implanted subcutaneously in both flanks of nude mice are represented (one way Anova *=p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 In vivo orthotopic implantations of ALK-wt, ALK-F1174L, and ALK-R1275Q cells generate undifferentiated NC cell-derived tumors A. Kaplan-Meier survival curves of orthotopically engrafted mice. Orthotopic tumor growth was followed by echography and mice were sacrificed once tumor on left AG reached approximately 800 mm 3 : JoMa1-ALK-F1174L, JoMa1-ALK-R1275Q, and JoMa1-ALK-wt group of mice were sacrificed from 24 to 35, 44 to 59, and 52 to 73 days post-injection, respectively (Gehan-Breslow-Wilcoxon test). All remaining mice were sacrificed 3 months post-injection. B. H&E and IHC staining for ALK and Ki67 of one representative orthotopic tumor of JoMa1-ALK-wt, -ALK-F1174L, and -ALK-R1275Q groups (magnification 40x, scale = 20 mum). C. IHC of various markers in one orthotopic JoMa1-ALK-F1174L tumor representative of all subcutaneous and orthotopic tumors (magnification 40x, scale = 20 mum). Positive controls for Th (ganglion), Phox2b (NB), CD99 (islet of Langerhans), Tyr (melanoma), and Ptprc (CD45) (spleen) are shown in small inserts. D. Bare graph showing expression levels of different genes in JoMa1-ALK tumors, NB murine transgenic model, JoMa1 cell lines, and adrenal glands.