**Author details**

*Immunohistochemistry - The Ageless Biotechnology*

the activity obtained with the BCIP/NBT reagent.

*scale, and in consequence blue deposits are visualized in black. Scale bar is 25 μm.*

technique is suitable for testing enzyme inhibitors.

The authors thank Tom Yohannan for language editing.

activity, Villamonte et al. [21]. License number: 4487101229606.

**3. Conclusions**

**Figure 6.**

**Acknowledgements**

**Conflict of interest**

of this book chapter.

antibodies used were mouse antihuman NTPDase1 primary antibody (clone BU-61, Ancell) and Alexa Fluor 488 goat anti-mouse secondary antibody (Thermo Fisher Scientific). Label is seen together with the lead precipitate in endothelium of blood vessels, especially abundant in the lamina propria of the mucosa layer, and in muscle cells, predominant in the muscular layer [21]. In **Figure 6**, the antibody against human placental-like alkaline phosphatase (PLAP; clone 8B6, Sigma-Aldrich) was used in Ishikawa cells to localize the protein by immunofluorescence, together with

*Placental-like alkaline phosphatase (PLAP) immunofluorescence (a) and in situ enzyme AP activity (b) in the Ishikawa endometrial carcinoma cell line. Nuclei are labeled with DAPI (c). Merge image (d) shows that precipitates are formed in cells expressing PLAP. Note that activity microphotograph (b) was obtained in gray* 

In conclusion, in situ histochemistry for ecto-nucleotidases is an easy-toperform, reproducible technique suitable for tissues and cells. The combined technique allows identification of the protein that has a precise enzyme activity. The

This study was supported by a grant from the Instituto de Salud Carlos III (FIS PI15/00036), co-funded by FEDER funds/European Regional Development Fund (ERDF)—"a Way to Build Europe"—/FONDOS FEDER "una manera de hacer Europa", and a grant from the Fundación Merck Salud (Ayuda Merck de Investigación 2016-Fertilidad). ARM was awarded a fellowship from the Asociación Española Contra el Cáncer (AECC). We thank CERCA Programme (Generalitat de Catalunya) for the institutional support. We are grateful to Serveis Científics I Tecnològics (Campus Bellvitge, Universitat de Barcelona) for the technical support.

**Figure 5** is reprinted by permission of Springer Nature Histochemistry and Cell Biology, Characterization of ecto-nucleotidases in human oviducts with an improved approach simultaneously identifying protein expression an *in situ* enzyme

The authors declare that there is no conflict of interest regarding the publication

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Mireia Martín-Satué\*, Aitor Rodríguez-Martínez and Carla Trapero Unit of Histology, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, University of Barcelona; Bellvitge Biomedical Research Institute (IDIBELL)—CIBERONC, Barcelona, Spain

\*Address all correspondence to: martinsatue@ub.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
