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ORIGINAL   DISTORTED  

For Research Use Only. Not For Use in Diagnostic Procedures  

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Growth and regeneration of the mammalian intestinal epithelium is driven primarily by a discrete population of stem cells, known as LGR5+ crypt base columnar (CBC) cells, resident at the base of the intestinal crypt. Despite their fundamental importance for intestinal development and regeneration, there is a lack of validated reagents that can discretely identify LGR5+ CBC cells and their direct progeny. This is likely due to the low abundance and/or accessibility of the LGR5 epitope. To address this problem, Cell Signaling Technology, Inc. (CST) has developed highly specific rabbit antibodies targeting Olfactomedin-4 (OLFM4), a glycoprotein that exhibits distinct expression in LGR5+ CBC cells in both human and mouse. These antibodies are able to detect human OLFM4, and mouse Olfm4, respectively, by Western blot in cell and tissue lysates, and by immunohistochemistry (IHC) in formalin-fixed paraffin-embedded tissue samples. Antibody specificity was confirmed by validation on cell types and/or tissues in which the presence/absence or relative abundance of Olfactomedin-4 protein has been described in the published literature. These antibody reagents provide a means to visualize CBC cells with exquisite sensitivity and specificity without the need for transgenic models. This will allow for greater insights into the role and regulation of CBC cells during development, regeneration and oncogenic transformation of the intestinal epithelium.  

ABSTRACT

METHODS

CONCLUSIONS

Antibody Validation

OLFM4 (D1E4M) XP® Rabbit mAb #14369 is a recombinant rabbit monoclonal antibody that detects human OLMF4 protein in western blot and immunohistochemistry with high specificity and sensitivity. Olfm4 (D6Y5A) XP® Rabbit mAb (Mouse Specific) #39141 is a recombinant rabbit monoclonal antibody that detects mouse Olfm4 protein in western blot, immunoprecipitation, immunofluorescence and immunohistochemistry with exceptional specificity. These antibodies may be used to detect or visualize OLFM4-expressing cells, including intestinal stem/progenitor cells and neutrophils, in normal tissues, intestinal adenoma / carcinoma, and in organoids derived from normal or cancerous tissues [1, 2]. For stem cell research, these tools provide a means to identify intestinal stem/progenitor cells without the obligatory use of transgenic mice models [3].

Antibody Validation

NEW  ANTIBODY  REAGENTS  TO  STUDY  STEM  CELL-­‐MEDIATED  DEVELOPMENT  AND  REGENERATION  OF  THE  MAMMALIAN  INTESTINAL  EPITHELIUM   Antony W. Wood, Richard Cotta, Katherine Crosby, Aparna Viswanathan, Christine Gagen and

Jessica Simendinger Cell Signaling Technology, Inc., Danvers MA 01923

Antony W. Wood, PhD email: antony.wood@cellsignal.com

www.cellsignal.com/######

Abstract  #1374  

Monoclonal Antibody Generation OLFM4 (D1E4M) XP® Rabbit mAb # 14369 and Olfm4 (D6Y5A) XP® Rabbit mAb (Mouse Specific) #39141 are recombinant rabbit monoclonal antibodies, generated at Cell Signaling Technology, Inc. using patented XMT® Technology. Western Blot Western blot analyses were performed using extracts from cell or tissue extracts, as described in the figure legends. Western blot protocol details can be found at http://www.cellsignal.com/common/content/content.jsp?id=western Immunohistochemistry Paraffin-embedded tissue sections were deparaffinized and rehydrated, then subjected to antigen retrieval in sodium citrate, pH 6.0. Primary antibodies were incubated overnight at 4°C. Detection was performed using SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 and SignalStain® DAB Substrate Kit #8059. Immunofluorescence Perfusion-fixed mouse small intestine was cryopreserved in 30% sucrose and embedded in OCT medium. 12 uM sections rinsed in PBS prior to blocking and permeabilization with 5% goat serum/PBS containing 0.3% Triton X-100 (PBST). Primary antibodies were diluted in PSBT containing 1% BSA and incubated overnight at 4°C. Detection was performed with Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 for 1 hour at 25C, and sections were counterstained with DyLight™ 554 Phalloidin #13054 and DRAQ5® #4084.

Fig.  1.  Messenger  RNA  expression  paBern  of  human  OLFM4  as  reported  in  BioGPS  (leI)  and  GTex  Portal  (right).    

Fig.   2.   Western   blot   analysis   of   extracts   from   human   small  intes)ne,  colon,  prostate,  and  selected  cancer  cell  lines  (OV-­‐90,  MCF7)  using  OLFM4  (D1E4M)  XP®  Rabbit  mAb  #14369  (upper)  and   β-­‐ac)n   (D6A8)   Rabbit   mAb   #8457   (lower).   Observed  protein   expression   is   consistent  with  mRNA   expression   levels  as   reported   in   bioinforma)c   databases   (BioGPS,   GTex   Portal,  Cancer  Cell  Line  Encyclopedia).    

Fig.  3.  Immunohistochemical  analysis  of  paraffin-­‐embedded  human  small  intes)ne  (leI)  and   human   colon   (right)   using   OLFM4   (D1E4M)   XP®   Rabbit   mAb   #14369.   Note   the  robust  expression  in  the  crypt  component  of  the  small  intes)ne.      

Fig.   4.   Immunohistochemical   analysis   of   paraffin-­‐embedded   human   colon   carcinoma,  Stage  I  (leI)  and  Stage  IV  (right)  using  OLFM4  (D1E4M)  XP®  Rabbit  mAb  #14369.  Note  the   stronger   expression   in   the   tumor  of   the   Stage   I   case   versus   the   Stage   IV   case,   as  reported  [4].  

OLFM4  (D1E4M)  XP®  Rabbit  mAb  #  14369   OLFM4  (D6Y5A)  XP®  Rabbit  mAb  (Mouse  Specific)  #39141  

Fig.   6.   Immunohistochemical  analysis   of   paraffin-­‐embedded  spleen   from   wild-­‐type   mouse  (upper)   and   Olfm4-­‐/-­‐   mouse  (lower),   using  Olfm4   (D6Y5A)  XP®  Rabbit   mAb   (Mouse   Specific)  #39141.    

Fig.  5.  Western  blot  analysis  of  extracts   from   mouse   small  intes)ne,   spleen,   and   colon  using   Olfm44   (D6Y5A)   XP®  Rabbit   mAb   #39141   (upper)  and  β-­‐ac)n  (D6A8)  Rabbit  mAb  #8457  (lower).  

Fig.  7.   Immunohistochemical   analysis  of  paraffin-­‐embedded  mouse  normal   small   intes)ne   (leI),  mouse  normal  colon  (middle)  and  Apc  (min/+)  mouse  intes)nal  adenoma,  using  Olfm4  DD6Y5A)  XP®  Rabbit  mAb  #39141.  Note  the  presence  of  strong  crypt  base  staining  in  small  intes)ne,  absence  of  staining  in  colon,  and  pervasive  staining  in  adenoma  (adjacent  to  normal  regions  with  crypt  base-­‐only  staining).  

Fig.   8.   Confocal   immunofluorescent   analysis   of   frozen   mouse   small-­‐intes)ne,  using  Olfm4   (D6Y5A)  XP®  Rabbit  mAb   (Mouse  Specific)  #39141  (green).   Ac)n   filaments   were   labeled   with   DyLightTM   554   Phalloidin  #13054  (red).  Blue  pseudocolor  =  DRAQ®  #4804  (fluorescent  dye).    

Mouse spleen tissues (wild-type and Olfm4-/-) were a generous gift from Dr. Griffin P. Rodgers, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

1.  van de Wetering M, et al. 2015. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161 (4): 933-945.

2.  Bayez et al. 2016. High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature 531: 53-58. 3.  Van der Flier et al., 2009. OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells.

Gastroenterology 137(1): 15-17. 4. Besson et al. 2011. A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new

nonmetastatic tumor marker. Mol. Cell. Proteomics 10 (12): M111.009712 DOI:10:1074/mcp.M111.009712.

Acknowledgements

References

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