**2.3 Combined immunolabeling and in situ nucleotidase activity experiments**

The technique uses the same tissue slide (or the same coverslip of cells) to identify both the activity, with in situ histochemistry (or cytochemistry), and the protein, with immunofluorescence (**Figure 4**) [21, 22]. Tissue sample sections or fixed cells grown on coverslips are washed twice with PBS and incubated with a blocking solution containing 20% normal goat serum and 0.2% gelatin in PBS at RT for 1 h and then incubated O/N at 4°C with the appropriate primary antibody. The samples are washed three times with PBS and twice with 50 mM Tris-maleate buffer. In situ nucleotidase activity experiment is performed as detailed previously, adding the appropriate nucleotide as substrate. Subsequently, the tissues are washed three times in PBS before incubating with the appropriate fluorescent-labeled secondary antibody. After three final washes with PBS, nuclei are labeled, and the samples mounted with aqueous mounting medium; a mounting medium containing DAPI can be used for this purpose (e.g., ProLong Gold antifade reagent with DAPI mounting medium from Thermo Fisher Scientific). The sections are observed and photographed under a Nikon Eclipse E800 Microscope. Pictures of bright field (for activity) and fluorescence (for protein immunolocalization and nuclei visualization) are taken sequentially from the same field.

We recommend that histochemistry be performed between primary and secondary antibody incubations, but other protocols are also feasible. This is of interest when using inhibitory antibodies. In these cases the in situ histochemistry should be performed at the beginning of the procedure. It also has to be taken into account that it might be necessary to test different nucleotide concentrations and incubation times in order to optimize the results for a particular tissue in order to minimize hampering of fluorescence capture by the dark brown lead deposits.

**Figures 5** and **6** are examples of this combined technique in tissue and cell culture, respectively. **Figure 5** shows immunofluorescence to localize NTPDase1, and in situ histochemistry for the ADPase activity in human fallopian tubes. The

#### **Figure 5.**

*Immunohistochemistry - The Ageless Biotechnology*

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**Figure 4.**

*activity detection protocols.*

*Scheme including the main steps of the method combining immunofluorescence and in situ ecto-nucleotidase* 

*Immunolocalization of NTPDase1 (a, d) and in situ ADPase histochemistry (b, e) in cryosections of human oviducts. NTPDase1 was detected with immunofluorescence in endothelial cells of lamina propria (a) and in smooth muscle cells (d). Microphotographs b and e show dark brown deposits corresponding to in situ ADPase activity. Merge images (c, f) confirmed that NTPDase1 is active in the same structures where it immunolocalizes. Reddish structure at top right of image is the blood inside the vessel. Scale bar is 25 μm. Reprinted by permission of springer nature histochemistry and cell biology, characterization of ectonucleotidases in human oviducts with an improved approach simultaneously identifying protein expression an in situ enzyme activity, Villamonte et al. [21].*

#### **Figure 6.**

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

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 the activity obtained with the BCIP/NBT reagent.

### **3. Conclusions**

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 technique is suitable for testing enzyme inhibitors.

### **Acknowledgements**

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. The authors thank Tom Yohannan for language editing.

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