Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Immunocytochemistry of mTRPM8 in transfected HEK-293 cells. ( A – F left) Confocal images of mTRPM8-EYFP transiently expressed in HEK-293 cells. EYFP (green), TRPM8 antibody (red) and Hoechst staining (blue). Merge images correspond to the overlap of the three fluorescent signals plus the bright field image. Scale bar: 30 µm. ( A – F right) Box plots represent the specificity ratio (SR) for each dilution of the respective antibody. Each dot corresponds to an individual EYFP+ cell. Box contains the 25th to 75th percentiles. Whiskers mark the 5th and 95th percentiles. The line inside the box denotes the median and the black dot represents the mean. (*** p < 0.001, Mann–Whitney test). ( G ) Bar histogram summarizing the SR mean ± SEM of each TRPM8 antibody and the controls without primary antibody (αRabbit or αMouse secondary antibodies alone). Dashed line indicates SR = 1. (*** p < 0.001, Kruskal–Wallis followed by Dunn’s post hoc test vs. αRabbit or αMouse). For each antibody and dilution, n > 270 cells; 4 fields from 2 independent transfections.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Immunocytochemistry, Transfection, Staining, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Western blot analysis for TRPM8 antibodies specificity. ( A – F ) TRPM8 immunoblots using ( A ) Alomone ( B ) ECM1, ( C ) Origene1, ( D ) ECM2, ( E ) ECM3 or ( F ) Origene2 antibodies. (−) lanes: untransfected HEK-293 cells, (+) lanes: HEK-293 cells transfected with mTRPM8-EYFP. Left: Immunoblot with each TRPM8 antibody. Right-top: EYFP immunoblotting on the same membrane. Right-bottom: GAPDH loading control. Black arrowheads indicate mTRPM8-EYFP bands revealed with antiTRPM8 antibody. Green arrowheads indicate mTRPM8-EYFP bands revealed with anti-GFP antibody. Red arrowheads indicate GAPDH bands. All blots were repeated at least 3 times to exclude a technical artefact when no anti-TRPM8 signal was observed. For each replicate, the same lysate was used for all antibodies.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Western Blot, Transfection, Membrane, Control

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Calcium imaging in DRG-cultured cells from Trpm8 BAC -EYFP + mice. ( A ) Representative traces of calcium transients evoked by mild cold (∼18–20 °C), WS-12 (10 µM) and KCl (30 mM) in EYFP+ and EYFP− neurons. Traces corresponding to individual cells are shown in grey. Colored traces represent averages of the respective individual traces. ( B ) Proportion of EYFP+ and EYFP− cells responding to cold and WS-12. ( C ) Representative immunocytochemistry performed after a calcium recording. Upper- and bottom-left: confocal images of the immunofluorescence. Green: EYFP. Red: TRPM8 (ECM1 antibody). Grey: βIII-Tubulin. Bottom-right: transmitted light and EYFP fluorescence from the same live cells before fixation. n = 432 cells from 2 mice. Scale bar: 50 µm.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Imaging, Cell Culture, Immunocytochemistry, Immunofluorescence, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Immunocytochemistry of endogenous TRPM8 in cultured DRG cells from the Trpm8 BAC -EYFP + mouse. ( A – F left) Confocal images of TRPM8+ sensory neurons. EYFP (green), TRPM8 antibody (red) and βIII-Tubulin (grey). Scale bar: 50 µm. ( A – F right) Box plots represent specificity ratio (SR) for each dilution of the respective antibody. Each dot corresponds to an individual EYFP+ cell. Box contains the 25th to 75th percentiles. Whiskers mark the 5th and 95th percentiles. The line inside the box denotes the median and the black dot represents the mean (* p < 0.05, ** p < 0.01, Mann–Whitney test). ( G ) Bar histogram summarizing the SR mean ± SEM of each TRPM8 antibody and the controls without primary antibody (αRabbit or αMouse secondary antibodies alone). Dashed line indicates SR = 1 (* p < 0.05, ** p < 0.01, Kruskal–Wallis followed by Dunn’s post hoc test vs. αRabbit or αMouse). n = at least 20 cells; 4 pictures from 2 mice for each antibody and dilution.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Immunocytochemistry, Cell Culture, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Immunohistochemistry of endogenous TRPM8 in DRG slices from the Trpm8 BAC -EYFP + mouse. ( A – F left) Confocal images of TRPM8+ sensory neurons. EYFP (green), TRPM8 antibody (red) and βIII-Tubulin (grey). Scale bar: 50 µm. ( A – F right) Box plots represent specificity ratio (SR) for each dilution of the respective antibody. Each dot corresponds to an individual EYFP+ cell. Box contains the 25th to 75th percentiles. Whiskers mark the 5th and 95th percentiles. The line inside the box denotes the median and the black dot represents the mean. (Differences among dilutions were not significant, Mann–Whitney test). ( G ) Bar histogram summarizing the SR mean ± SEM of each TRPM8 antibody and the controls without primary antibody (αRabbit or αMouse secondary antibodies alone). Dashed line indicates SR = 1. (* p < 0.05, ** p < 0.01, Kruskal–Wallis followed by Dunn´s post hoc test vs. αRabbit or αMouse). n = at least 18 cells; 4 images from 2 mice for each antibody and dilution.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Immunohistochemistry, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Immunofluorescence of endogenous TRPM8 in DRG cells and slices from the TRPM8 KO mouse. ( A , C ) Immunocytochemistry. ( B , D ) Immunohistochemistry. ( A – D left) Confocal images of TRPM8 KO ( Trpm8 EGFPf ; B6;129S1(FVB)- Trpm8 tm1Apat /J) sensory neurons. EGFP (green), TRPM8 antibody (red) and βIII-Tubulin (grey). Scale bar: 50 µm. ( A – D right) Box plots represent specificity ratio (SR) for each dilution of the respective antibody. Each dot corresponds to an individual EGFP+ cell. Box contains the 25th to 75th percentiles. Whiskers mark the 5th and 95th percentiles. The line inside the box denotes the median and the black dot represents the mean (** p < 0.01, Mann–Whitney test). ( E,F ) Bar histograms summarizing the SR mean ± SEM of each TRPM8 antibody in the KO and the reporter ( Trpm8 BAC -EYFP + , named M8-EYFP in the figure) mouse in ICC ( E ) and IHC ( F ). Dashed line indicates mean SR for the control without primary antibody (** p < 0.01, Mann–Whitney test). n = at least 22 cells; 4 pictures from 2 mice for each antibody and dilution.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Immunofluorescence, Immunocytochemistry, Immunohistochemistry, MANN-WHITNEY, Control

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Antibody performance with the different techniques used in this study. − poor, + regular, ++ good, +++ excellent.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: TRPM8 , Origene1 , OriGene , TA307827 , Rabbit , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Isolation

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Immunohistochemistry using anti-TRPM8 in pancreatic tissues. ( A ) Normal pancreatic tissues. ( B ) Chronic pancreatitis. ( C ) Pancreatic intra-epithelial neoplasm. ( D ) Intraductal papillary mucinous neoplasm.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Immunohistochemistry

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Immunohistochemical analysis of TRPM8 in malignant pancreatic tumors. ( A , B ) Adenocarcinoma. ( C , D ) Adenosquamous carcinoma. ( E , F ) Solid pseudo-papillary neoplasm ( G , H ) Acinar cell carcinoma. ( I , J ) Neuroendocrine tumor. ( A , C , E , G , I ) H and E, original magnification ×200. ( B , D , F , H , J ) Immunohistochemistry using anti-TRPM8 antibodies, original magnification ×400.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Immunohistochemical staining, Immunohistochemistry

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Expression of TRPM8 in various types of histopathology in pancreatic tumors with regard to the intensity of immunoreactivity and the percentage of positive cells. The total number of cases examined is 308. The values represent the number of specimens with the corresponding proportions in parentheses.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Expressing, Histopathology

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Expression levels of TRPM8 in pancreatic adenocarcinoma. Images in ( A , C , E ) (original magnification ×200) represent the H&E sections of the same tumor as the TRPM8 immunohistochemical staining in ( B , D , F ) (original magnification ×400), respectively. Expression levels of TRPM8: ( B ) no-to-low; ( D ) moderate; ( F ) high.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Anti-TRPM8 immunoreactivity in pancreatic adenocarcinoma positively correlates with primary tumor size and stages. The distribution of the IHC score for expression of TRPM8 is displayed against age/gender, histological grade, primary tumor (T), and stage. The total number of specimens of pancreatic adenocarcinoma examined is 280. Polyserial correlation between the primary tumor size (T) or the tumor stages and the log-transformed IHC score is 0.13 and 0.10, respectively.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Expressing, Transformation Assay

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: The percent coverage ± standard error for expression of TRPM8 in pancreatic adenocarcinoma with respect to the intensity and the tumor stage. The numbers in parentheses represent the patient counts. NA, not applicable.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Expressing

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Over-expression of TRPM8 protein in human pancreatic adenocarcinoma cell lines. ( A ) The protein levels of TRPM8 in H6c7, MIA PaCa-2, PANC-1, and BxPC-3 cells were determined using immunoblotting with anti-TRPM8 antibodies. The GAPDH protein levels were analyzed as internal controls. ( B ) The relative protein levels of TRPM8 are expressed as % (mean ± standard error) of that in H6c7.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: Over Expression, Western Blot

Journal: Cells

Article Title: Aberrantly Over-Expressed TRPM8 Channels in Pancreatic Adenocarcinoma: Correlation with Tumor Size/Stage and Requirement for Cancer Cells Invasion

doi: 10.3390/cells3020500

Figure Lengend Snippet: Short hairpin RNA-mediated silencing of TRPM8 impaired invasion of pancreatic adenocarcinoma cells. BxPC-3 and MIA PaCa-2 cells transfected with either anti- TRPM8 shRNA or NC shRNA were analyzed for cell invasion using the trans-well assay. Cell invasion is expressed % (mean ± standard error) of that in the cells transfected with control shRNA. Representative images of the invaded cells stained with crystal violet are shown at 200× magnification.

Article Snippet: The membrane was blocked in 5% non-fat dry milk and incubated overnight at 4 °C with anti-human TRPM8 (rabbit polyclonal) or anti-human GAPDH (mouse monoclonal) antibodies (OriGene Technologies, Rockville, MD, USA) used at 1:750 and 1:2500 dilution, respectively.

Techniques: shRNA, Transfection, Staining