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VPAC2 receptor expression in human normal and neoplastic tissues: evaluation of the novel MAB SP235.

Schulz S, Mann A, Novakhov B, Piggins HD, Lupp A - Endocr Connect (2014)

Bottom Line: SP235 immunohistochemistry detected VPAC2 receptors in lymphocytes present in spleen, tonsils, lymph nodes and Peyer's patches, chief cells of gastric mucosa, exocrine and endocrine pancreas, kidney tubules and blood vessels.In addition, VPAC2 was observed in thyroid, gastric and lung carcinomas, pancreatic adenocarcinomas, sarcomas and neuroendocrine tumours.SP235 may prove of great value in the identification of VPAC2 receptors during routine histopathological examination.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and ToxicologyJena University Hospital, Friedrich Schiller University Jena, Drackendorfer Straße 1, D-07747 Jena, GermanyFaculty of Life SciencesUniversity of Manchester, Manchester M13 9PT, UK Stefan.Schulz@med.uni-jena.de.

No MeSH data available.


Related in: MedlinePlus

SP235 immunohistochemistry of human normal and neoplastic tissues. Sections were dewaxed, microwaved in citric acid and incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in AEC and lightly counterstained with haematoxylin. Insets in A and D, for adsorption controls the SP235 was incubated with 10 μg/ml of the peptide used for immunisations (+ peptide). Scale bar, A=B=C=F=I=250 μm and D=E=G=H=100 μm.
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fig3: SP235 immunohistochemistry of human normal and neoplastic tissues. Sections were dewaxed, microwaved in citric acid and incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in AEC and lightly counterstained with haematoxylin. Insets in A and D, for adsorption controls the SP235 was incubated with 10 μg/ml of the peptide used for immunisations (+ peptide). Scale bar, A=B=C=F=I=250 μm and D=E=G=H=100 μm.

Mentions: Specificity of the novel rabbit monoclonal anti-VPAC2 antibody (SP235) was monitored using western blotting analysis. When extracts from stably transfected HEK 293 cells were electrophoretically separated and blotted onto PVDF membranes, SP235 detected a broad band migrating at 50–70 kDa in cells stably expressing the VPAC2 but not in cells expressing the VPAC1 receptor (Fig. 1A, left panel). Conversely, the rabbit monoclonal anti-VPAC1 antibody (SP234) detected a broad band migrating at 55–75 kDa in cells stably expressing the VPAC1 receptor but not in cells expressing the VPAC2 receptor (Fig. 1A, right panel). SP235 was further characterised using immunofluorescent staining of transfected cells. As depicted in Fig. 1B, the anti-VPAC2 antibody (SP235) revealed prominent immunofluorescence localised at the plasma membrane only in VPAC2- but not in VPAC1-expressing cells. Conversely, the anti-VPAC1 antibody (SP234) revealed prominent immunofluorescence localised at the plasma membrane only in VPAC1, but not in VPAC2-expressing cells (Fig. 1B). Next, SP235 was tested for possible cross-reactivity with other proteins present in the extracts from mouse, rat or human tissues. A comparison of the carboxyl-terminal sequences revealed a high degree of homology between mouse, rat and human VPAC2 receptors (Fig. 2A). Consequently, cross-reactivity of SP235 to both mouse and rat VPAC2 receptors was observed (Fig. 2B and C). When extracts from a variety of mouse tissues were electrophoretically separated and blotted onto PVDF membranes, the anti-VPAC2 antibody (SP235) revealed a broad receptor-like band with a molecular weight similar to that observed for recombinant VPAC2 receptor in gastric mucosa, pancreas and brain from WT (C57BL6) but not from Vpac2-knock-out mice (Fig. 2B). In addition, SP235 revealed faint bands of similar size in large intestine and spleen, but not in small intestine or testis (Fig. 2B). Immunoreactive bands for SP235 were completely abolished by preabsorbtion with 10 μg/ml of its immunising peptide (Supplementary Figure S1, see section on supplementary data given at the end of this article). The anti-VPAC2 antibody (SP235) was then subjected to immunohistochemical staining of a selection of mouse, rat and human tissues revealing similar cellular and subcellular localisations of VPAC2 receptors in all three species. As depicted in Fig. 2C, VPAC2 receptors were detected at the plasma membrane of chief cells in the gastric mucosa and the basal membrane of proximal kidney tubules of both rats and humans. In addition, tissue immunostaining of SP235 was completely abolished by preadsorption with its immunising peptide (Fig. 3A and D).


VPAC2 receptor expression in human normal and neoplastic tissues: evaluation of the novel MAB SP235.

Schulz S, Mann A, Novakhov B, Piggins HD, Lupp A - Endocr Connect (2014)

SP235 immunohistochemistry of human normal and neoplastic tissues. Sections were dewaxed, microwaved in citric acid and incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in AEC and lightly counterstained with haematoxylin. Insets in A and D, for adsorption controls the SP235 was incubated with 10 μg/ml of the peptide used for immunisations (+ peptide). Scale bar, A=B=C=F=I=250 μm and D=E=G=H=100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: SP235 immunohistochemistry of human normal and neoplastic tissues. Sections were dewaxed, microwaved in citric acid and incubated with the rabbit monoclonal anti-VPAC2 antibody (SP235) at a dilution of 1:500. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in AEC and lightly counterstained with haematoxylin. Insets in A and D, for adsorption controls the SP235 was incubated with 10 μg/ml of the peptide used for immunisations (+ peptide). Scale bar, A=B=C=F=I=250 μm and D=E=G=H=100 μm.
Mentions: Specificity of the novel rabbit monoclonal anti-VPAC2 antibody (SP235) was monitored using western blotting analysis. When extracts from stably transfected HEK 293 cells were electrophoretically separated and blotted onto PVDF membranes, SP235 detected a broad band migrating at 50–70 kDa in cells stably expressing the VPAC2 but not in cells expressing the VPAC1 receptor (Fig. 1A, left panel). Conversely, the rabbit monoclonal anti-VPAC1 antibody (SP234) detected a broad band migrating at 55–75 kDa in cells stably expressing the VPAC1 receptor but not in cells expressing the VPAC2 receptor (Fig. 1A, right panel). SP235 was further characterised using immunofluorescent staining of transfected cells. As depicted in Fig. 1B, the anti-VPAC2 antibody (SP235) revealed prominent immunofluorescence localised at the plasma membrane only in VPAC2- but not in VPAC1-expressing cells. Conversely, the anti-VPAC1 antibody (SP234) revealed prominent immunofluorescence localised at the plasma membrane only in VPAC1, but not in VPAC2-expressing cells (Fig. 1B). Next, SP235 was tested for possible cross-reactivity with other proteins present in the extracts from mouse, rat or human tissues. A comparison of the carboxyl-terminal sequences revealed a high degree of homology between mouse, rat and human VPAC2 receptors (Fig. 2A). Consequently, cross-reactivity of SP235 to both mouse and rat VPAC2 receptors was observed (Fig. 2B and C). When extracts from a variety of mouse tissues were electrophoretically separated and blotted onto PVDF membranes, the anti-VPAC2 antibody (SP235) revealed a broad receptor-like band with a molecular weight similar to that observed for recombinant VPAC2 receptor in gastric mucosa, pancreas and brain from WT (C57BL6) but not from Vpac2-knock-out mice (Fig. 2B). In addition, SP235 revealed faint bands of similar size in large intestine and spleen, but not in small intestine or testis (Fig. 2B). Immunoreactive bands for SP235 were completely abolished by preabsorbtion with 10 μg/ml of its immunising peptide (Supplementary Figure S1, see section on supplementary data given at the end of this article). The anti-VPAC2 antibody (SP235) was then subjected to immunohistochemical staining of a selection of mouse, rat and human tissues revealing similar cellular and subcellular localisations of VPAC2 receptors in all three species. As depicted in Fig. 2C, VPAC2 receptors were detected at the plasma membrane of chief cells in the gastric mucosa and the basal membrane of proximal kidney tubules of both rats and humans. In addition, tissue immunostaining of SP235 was completely abolished by preadsorption with its immunising peptide (Fig. 3A and D).

Bottom Line: SP235 immunohistochemistry detected VPAC2 receptors in lymphocytes present in spleen, tonsils, lymph nodes and Peyer's patches, chief cells of gastric mucosa, exocrine and endocrine pancreas, kidney tubules and blood vessels.In addition, VPAC2 was observed in thyroid, gastric and lung carcinomas, pancreatic adenocarcinomas, sarcomas and neuroendocrine tumours.SP235 may prove of great value in the identification of VPAC2 receptors during routine histopathological examination.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and ToxicologyJena University Hospital, Friedrich Schiller University Jena, Drackendorfer Straße 1, D-07747 Jena, GermanyFaculty of Life SciencesUniversity of Manchester, Manchester M13 9PT, UK Stefan.Schulz@med.uni-jena.de.

No MeSH data available.


Related in: MedlinePlus