rabbit anti cftr  (Alomone Labs)

Journal: EMBO Molecular Medicine

Article Title: Benchmarking porcine pancreatic ductal organoids for drug screening applications

doi: 10.1038/s44321-025-00330-3

Figure Lengend Snippet: ( A ) Schema showing the process for generating PPDO. ( B – E ) Brightfield microscopy images of early passage PPDO and HPDO from Em- ( B ), EPN ( C ), LPN ( D ) and from a human donor ( E ). Scale bar: 500 µm. ( B’ – E’ ) Brightfield microscopy images of late passage PPDO and HPDO from Em ( B’ ), EPN ( C’ ), LPN ( D’ ) and from a human donor ( E ). Scale bar: 500 µm. ( F – H ) Single-plane confocal images of PPDO derived from EPN pig pancreas immunostained against CFTR-Phalloidin-PDX1 ( F ), SOX9-GP2-NKX6-1/GCG ( G ), KRT7-CDH1 ( H ) and counterstained with DAPI. Insets show the individual channels of the merge image. PPDO were stained at passage 3. Scale bar: 50 µm. ( I – K ) Brightfield microscopy images of early passage Em PPDO at the beginning of the live imaging, treated with DMSO ( I ), forskolin ( J ), and secretin ( K ). Scale bar 500 µm. ( I’ –K’ ) Brightfield microscopy images of early passage Em PPDO at the end of the live imaging, treated with DMSO ( I’ ), forskolin ( J’ ) and secretin ( K’ ). Scale bar 500 µm. ( L ) Quantification of the lumen area expansion of PPDO following 4 h of live imaging and treatments with forskolin and secretin. n = 3 independent experiments with different PPDO lines (1 Em, 1 EPN and 1 Ad). Data are shown as mean ± SD. One-way ANOVA followed by Dunnett’s multiple comparisons test was used to assess significance. P val are: * Padj = 0.015 for DMSO vs forskolin and * Padj = 0.0140 for DMSO vs secretin. ( M ) Quantification of the lumen area expansion of HPDO following 4 h of live imaging and treatment with forskolin. n = 4 independent experiments with 4 HPDO lines. Data are shown as mean ± SD. Mann–Whitney test was used to assess significance. * P = 0.0286. .

Article Snippet: Primary antibodies used in this study were: a-SOX9 (rabbit, 1:800, Millipore AB3555), a-KRT5 (rabbit, 1:200, abcam ab53121), a-KRT7 (mouse, 1:200, Agilent Technologies M701829-2), a-KRT8/18 (guinea pig, 1:1000, Origene BP5007), a-PAN-KRT (rabbit, 1:500, Agilent Z0622), a-BMPR1A (mouse, 1:400, LSBio LS-C191759), a-CDH1 (mouse, 1:800, BD Biosciences 610181), a-CDH1 (rat, 1:500, Takara M108), a-CFTR (rabbit, 1:100, Cell Signaling 78335), a-GP2 (mouse, 1:100, MBL D277-3), a-PDX1 (goat, 1:200, R&D Systems AF2419), a-GCG (mouse, 1:200, Sigma-Aldrich G2654), a-AGR2 (rabbit, 1:200, Cell Signaling 13062S), a-GCG (guinea pig, 1:800, Takara M182), a-NKX6-1 (mouse, 1:100, Developmental Studies Hybridoma Bank F55A10), a-NEUROG3 (sheep, R&D Systems AF3444), a-Pancreatic amylase (rabbit, 1:200, abcam 21156), a-INS (guinea pig, 1:200, LSBio (BIOZOL), LS-C85862-1), a-GCG (guinea pig, 1:1000, Takara, M182), Phalloidin Alexa Fluor 546 (Invitrogen A22283), a-cleaved-caspase3-ASP175 (1:200, Cell Signaling, 9661S), a-Ki67-Vio G570 (1:100, Miltenyi Biotec, 130-133-796).

Techniques: Microscopy, Derivative Assay, Staining, Imaging, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Benchmarking porcine pancreatic ductal organoids for drug screening applications

doi: 10.1038/s44321-025-00330-3

Figure Lengend Snippet: ( A – C ) Single-plane confocal images of PPDO derived from one Em pig pancreas immunostained against Pan-Cytokeratin (PAN-CK)-BMPR1A-CDH1 ( A ), SOX9-Phalloidin-NKX6-1 ( B ), CFTR-GP2-PDX1 ( C ) and counterstained with DAPI. Insets show the individual channels of the merge image. PPDO were stained at passage 2. Scale bar: 50 µm. ( D –F ) Single-plane confocal images of PPDO derived from an LPN pig pancreas immunostained against KRT5-BMPR1A-CDH1 ( D ), SOX9-GCG/NKX6-1-Phalloidin ( E ), CFTR-GP2-PDX1 ( F ) and counterstained with DAPI. Insets show the individual channels of the merge image. PPDO were stained at passage 4. Scale bar: 50 µm. ( G – I ) Single-plane confocal images of PPDO derived from an Ad pig pancreas immunostained against CFTR-GP2-PDX1 ( G ), SOX9-Phalloidin-CDH1 ( H ), AMY3A-KRT7-NEUROG3 ( I ), and counterstained with DAPI. PPDO were stained at passage 3. Insets show the individual channels of the merge image. Scale bar: 50 µm. ( J –L ) Single-plane confocal images of HPDO immunostained against KRT7-AGR2-CDH1 ( J ), CFTR-BMPR1A-NKX6.1 ( K ), SOX9-Phalloidin-PDX1 ( L ), and counterstained with DAPI. Insets show the individual channels of the merge image. HPDO were stained at passage 3. Scale bar: 50 µm. ( M , N ) Single-plane confocal images of porcine pancreas cryosections (Ad) immunostained against NKX6.1-SOX9-CDH1 ( M ) and phalloidin-555-PDX1 ( N ), and counterstained with DAPI. Scale bar: 50 µm. ( O ) Quantification of the organoid area of late passage (>5) PPDO following 4 h of live imaging and treatment with forskolin. n = 3 independent experiments with 3 PPDO lines (2 EPN and 1 Ad). Data are shown as mean ± SD. One-way ANOVA followed by Dunnett’s multiple comparisons test was used to assess significance. Not-significant (ns) P = 0.9010.

Article Snippet: Primary antibodies used in this study were: a-SOX9 (rabbit, 1:800, Millipore AB3555), a-KRT5 (rabbit, 1:200, abcam ab53121), a-KRT7 (mouse, 1:200, Agilent Technologies M701829-2), a-KRT8/18 (guinea pig, 1:1000, Origene BP5007), a-PAN-KRT (rabbit, 1:500, Agilent Z0622), a-BMPR1A (mouse, 1:400, LSBio LS-C191759), a-CDH1 (mouse, 1:800, BD Biosciences 610181), a-CDH1 (rat, 1:500, Takara M108), a-CFTR (rabbit, 1:100, Cell Signaling 78335), a-GP2 (mouse, 1:100, MBL D277-3), a-PDX1 (goat, 1:200, R&D Systems AF2419), a-GCG (mouse, 1:200, Sigma-Aldrich G2654), a-AGR2 (rabbit, 1:200, Cell Signaling 13062S), a-GCG (guinea pig, 1:800, Takara M182), a-NKX6-1 (mouse, 1:100, Developmental Studies Hybridoma Bank F55A10), a-NEUROG3 (sheep, R&D Systems AF3444), a-Pancreatic amylase (rabbit, 1:200, abcam 21156), a-INS (guinea pig, 1:200, LSBio (BIOZOL), LS-C85862-1), a-GCG (guinea pig, 1:1000, Takara, M182), Phalloidin Alexa Fluor 546 (Invitrogen A22283), a-cleaved-caspase3-ASP175 (1:200, Cell Signaling, 9661S), a-Ki67-Vio G570 (1:100, Miltenyi Biotec, 130-133-796).

Techniques: Derivative Assay, Staining, Imaging

Journal: EMBO Molecular Medicine

Article Title: Benchmarking porcine pancreatic ductal organoids for drug screening applications

doi: 10.1038/s44321-025-00330-3

Figure Lengend Snippet: ( A , B ) Brightfield microscopy images of PPDO in complete ( A ) or porcine serum supplemented ( B ). Scale bar 500 µm. ( C , D ) Brightfield microscopy images of PPDO at the end of differentiation in complete ( C ) or at the end of the differentiation using S5 + S6 combination ( D ) media. Scale bar 500 µm. ( E –J ) Bar plots showing the fold change of gene expression analysis at the end of the differentiation after treatment with porcine serum. Gene expression was measured for CFTR ( E ), KRT7 ( F ), NEUROD1 ( G ), GCG ( H ), INS ( I ), and SST ( J ). n = 3 independent PPDO lines (1 Em and 2 EPN). Absence of samples from the plots indicate non-detectable amplification following the RT-PCR. Data are shown as mean ± SEM. Unpaired Student’s t-test was used to assess significance with * P = 0.0196 for NEUROD1 . ( K , L ) Single-plane confocal images of PPDO in growth media ( K ) or in differentiation media from Loomans et al ( L ) (see Methods). PPDO were immunostained against INS-KRT7-CHGA and counterstained with DAPI. Experiment was repeated with n = 3 biological replicates (1 Em-1 EPN-1 Ad). Scale bar: 50 µm. ( M , N ) Brightfield microscopy images of HPDO in differentiation media ( M ) or differentiation media supplemented with DAPT/DEAB small molecules ( N ). Experiment was repeated with n = 3 biological replicates. Scale bar 500 µm.

Article Snippet: Primary antibodies used in this study were: a-SOX9 (rabbit, 1:800, Millipore AB3555), a-KRT5 (rabbit, 1:200, abcam ab53121), a-KRT7 (mouse, 1:200, Agilent Technologies M701829-2), a-KRT8/18 (guinea pig, 1:1000, Origene BP5007), a-PAN-KRT (rabbit, 1:500, Agilent Z0622), a-BMPR1A (mouse, 1:400, LSBio LS-C191759), a-CDH1 (mouse, 1:800, BD Biosciences 610181), a-CDH1 (rat, 1:500, Takara M108), a-CFTR (rabbit, 1:100, Cell Signaling 78335), a-GP2 (mouse, 1:100, MBL D277-3), a-PDX1 (goat, 1:200, R&D Systems AF2419), a-GCG (mouse, 1:200, Sigma-Aldrich G2654), a-AGR2 (rabbit, 1:200, Cell Signaling 13062S), a-GCG (guinea pig, 1:800, Takara M182), a-NKX6-1 (mouse, 1:100, Developmental Studies Hybridoma Bank F55A10), a-NEUROG3 (sheep, R&D Systems AF3444), a-Pancreatic amylase (rabbit, 1:200, abcam 21156), a-INS (guinea pig, 1:200, LSBio (BIOZOL), LS-C85862-1), a-GCG (guinea pig, 1:1000, Takara, M182), Phalloidin Alexa Fluor 546 (Invitrogen A22283), a-cleaved-caspase3-ASP175 (1:200, Cell Signaling, 9661S), a-Ki67-Vio G570 (1:100, Miltenyi Biotec, 130-133-796).

Techniques: Microscopy, Gene Expression, Amplification, Reverse Transcription Polymerase Chain Reaction