Cryostat section were 10 µm thick. The primary antibodies used were: guinea pig anti-Pdx1 (1/750; C.W. Wright), guinea pig anti-porcine insulin (1/400; DAKO, A0564), rabbit anti-insulin (1/3000; Molecular Probes, 701265), mouse anti-glucagon (1/1000; Sigma, G2654), rabbit anti-glucagon (1/200; DAKO, A0565), mouse anti-somatostatin (1/200; BCBC Ab1985), rabbit anti-somatostatin (1/200; DAKO, A0566), goat anti-somatostatin (1/200; Santa Cruz Biotechnology, sc-55565), rabbit anti-GFP (1/400; Molecular Probes, A11122), chicken anti-GFP (1/500; Abcam, ab13970), mouse anti-Ppy (1/200; Y. Fujitani)70 (link),mouse anti-Ppy (1/1000; R&D Biosystems, MAB62971), mouse anti-Pyy (1/1000; Abcam, ab112474), rabbit anti-Chga (1/200; Abcam, ab68271) and rabbit anti-Iapp (1/500; Abcam, ab254259). Secondary antibodies were coupled to Alexa 488, 405, 568, 647 (1/500; Molecular Probes) or TRITC, FITC, Cy3 and Cy5 (1/500; Southern Biotech). All antibodies are listed in Supplementary Table 2 . Sections were also stained with DAPI. All sections were examined with a confocal microscope (Leica TCS SPE).
Ab68271
Manufactured by Abcam
Sourced in United Kingdom
Ab68271 is a primary antibody that recognizes the IL-1 alpha protein. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab112474, by Abcam (1 mentions)
A11122, by Abcam (1 mentions)
A0564, by Agilent Technologies (1 mentions)
Tcs spe, by Leica (1 mentions)
A0565, by Agilent Technologies (1 mentions)
A0566, by Agilent Technologies (1 mentions)
Ab13970, by Abcam (1 mentions)
Ab109199, by Abcam (1 mentions)
Ab32034, by Abcam (1 mentions)
Ab32127, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Ab180943, by Abcam (1 mentions)
Anti mycn, by Cell Signaling Technology (1 mentions)
Anti sox11, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab134152, by Abcam (1 mentions)
Flex ihc microscope slides, by Agilent Technologies (1 mentions)
Scn400, by Leica (1 mentions)
Ab16659, by Abcam (1 mentions)
Autostainer link, by Agilent Technologies (1 mentions)
4 protocols using ab68271
1
Comprehensive Pancreatic Tissue Immunolabeling
2
Protein Expression Analysis in Cells
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Total protein was lysed from cells using radioimmunoprecipitation assay buffer (RIPA buffer; Beyotime, Shanghai, China) supplemented with protease inhibitors. SDS/PAGE was used to separate 30 µg of total proteins, which were then transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk and then incubated overnight at 4°C with specific primary antibodies. The membranes were washed and incubated with secondary antibodies at room temperature for 1 h. Electro chemiluminescent detection was used to visualize the protein bands. The expressions of GAPDH and β-actin were used as internal controls. The primary antibodies were used in this study: anti-SOX11 (Santa Cruz, CA, United States), anti-MYCN (#84406, Cell Signaling Technology, United States), anti-p21 (ab109199, Abcam, United States), anti-p27 (ab32034, Abcam, United States), anti-Cyclin D1 (ab134175, Abcam, United States), anti-NSE (ab180943, Abcam, United States), anti-CHGA (ab68271, Abcam, United States), and anti-SYP (ab32127, Abcam, United States). Quantification of the western blots was performed using ImageJ.
3
Immunohistochemical Analysis of Neuroendocrine Tumors
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The resected tumor tissues, formalin-fixed and paraffin-embedded in tissue blocks (FFPE), were sectioned in 4 μm slices and put on FLEX IHC microscope slides (Dako, Stockholm, Sweden). The sections were then deparaffinized, rehydrated, and processed with Dako EnVisionTM FLEX antigen retrieval EDTA buffer pH 9 using the DAKO PT Link module (PT Link, DakoCytomation, Glostrup, Denmark) according to the manufacturer’s instructions. The staining was performed in an autostainer (DAKO Autotstainer Plus, DakoCytomation, Glostrup, Denmark) according to manufacturer’s instructions. A human small intestinal NET was used as a positive control for somatostatin receptor 2 (SSTR2) and chromogranin A (CgA). Applied antibodies were rabbit anti-SSTR2 (ab134152, 1:50; Abcam, Cambridge, UK) and rabbit anti-CgA (ab68271, 1:500; Abcam, Cambridge, UK). The stained tissue sections were reviewed and assessed by two certified pathologists. Images for the article were digitalized using a Leica SCN400 and SlidePath Gateway Client LAN 2b4 (Leica Microsystems, Buffalo Grove, IL, USA).
4
Immunohistochemical Characterization of MTC Tumours
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The formalin-fixed tumours were dehydrated, embedded in paraffin, and sliced into sections of 4 μm, according to standard procedures. To verify the MTC origin of the tumours, sections from the mock-treated control group were stained with haematoxylin and eosin (H&E) for morphological examination, and by using antibodies for MTC markers chromogranin A (CgA, dilution 1:500; ab68271, Abcam, Cambridge, England), synaptophysin (Syn, dilution 1:25; ab16659, Abcam), and calcitonin (Ctn, dilution 1:1000; A0576, Dako, Glostrup, Denmark). Antibodies were incubated for 1 hour.
For the immunohistochemical (IHC) staining, tumour sections were collected on glass slides and then treated with EnVision™ FLEX Target Retrieval Solution (high pH) using a PT-Link (Dako). The staining was done in an Autostainer Link using EnVision™ FLEX (Dako) according to the manufacturer’s instructions. Positive and negative controls were included in each run. A microscope (20x magnification, Eclipse E1000, Nikon Instruments, Amsterdam, Netherlands) equipped with a camera (ProgRes C7, Jenoptik, Jena, Germany) was used for imaging of the stained tumour sections.
For the immunohistochemical (IHC) staining, tumour sections were collected on glass slides and then treated with EnVision™ FLEX Target Retrieval Solution (high pH) using a PT-Link (Dako). The staining was done in an Autostainer Link using EnVision™ FLEX (Dako) according to the manufacturer’s instructions. Positive and negative controls were included in each run. A microscope (20x magnification, Eclipse E1000, Nikon Instruments, Amsterdam, Netherlands) equipped with a camera (ProgRes C7, Jenoptik, Jena, Germany) was used for imaging of the stained tumour sections.
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