**2. In situ nucleotidase activity experiments**

The protocol detailed here for the detection of E-NTPDases, E-NPPs, and eN is based on the Wachstein-Meisel lead phosphate method [18], and the protocol for AP identification is based on the Gossrau method [19], with some modifications.

#### **2.1 Wachstein-Meisel lead phosphate-based method**

#### *2.1.1 In tissue samples*

Tissue pieces are fixed with 4% paraformaldehyde for a time period varying from a few hours to 2 days depending on the size (**Figure 2**). Following fixation, tissue pieces are cryopreserved by embedding them in 30% sucrose (in Milli-Q H2O) O/N or until tissue sinks. Tissues are then embedded in optimum cutting temperature (OCT) compound and cut with a cryostat into 15 μm-thick sections that are put on slides. It is recommended that pretreated slides be used, either homemade polylysine-treated or the commercial ones, to eliminate tissue loss during the procedure. Sections are stored at −20°C until use.

Tissue slides are rinsed with phosphate buffered saline (PBS) to remove OCT compound and washed twice with 50 mM Tris-maleate buffer pH 7.4 at RT. Slides are then incubated for 30 min at RT with preincubation buffer (50 mM Tris-maleate buffer pH 7.4 containing 2 mM MgCl2 and 250 mM sucrose) and then for 1 h at 37°C with the enzyme reaction buffer (50 mM Tris-maleate buffer pH 7.4 supplemented with 250 mM sucrose, 2 mM MgCl2, 5 mM MnCl2, 2 mM Pb(NO3)2, and 2 mM

**119**

**Figure 2.**

*In Situ Identification of Ectoenzymes Involved in the Hydrolysis of Extracellular Nucleotides*

*Scheme including the main steps of the lead phosphate-based method for ecto-nucleotidase activity detection.*

*DOI: http://dx.doi.org/10.5772/intechopen.84495*

*In Situ Identification of Ectoenzymes Involved in the Hydrolysis of Extracellular Nucleotides DOI: http://dx.doi.org/10.5772/intechopen.84495*

**Figure 2.**

*Immunohistochemistry - The Ageless Biotechnology*

found in the liver, bone, and kidneys [15].

significantly influence purinergic signaling [17].

**2. In situ nucleotidase activity experiments**

**2.1 Wachstein-Meisel lead phosphate-based method**

procedure. Sections are stored at −20°C until use.

*2.1.1 In tissue samples*

APs are zinc-containing dimeric membrane-bound glycoproteins that require magnesium ion for the hydrolysis of a wide range of phosphomonoesters. Although optimum activity occurs at alkaline pH (9.3–10.3), they are also active at a physiological pH, and they are primarily responsible for the PPi phosphohydrolysis in neutral and alkaline environments. APs are classified by their tissue expression and distribution [14]; in humans there are four types of APs in two main groups: the tissue nonspecific alkaline phosphatase (TNAP), with only one member, and the tissue-specific APs, which include the placental-like alkaline phosphatase (PLAP), the germ cell alkaline phosphatase (GCAP), and the intestinal alkaline phosphatase (IAP). Despite the fact that TNAP expression is not tissue-specific, it is mainly

APs catalyze the hydrolysis of monoesters of phosphoric acids and have extensive substrate specificity in vitro. For example, TNAP is able to hydrolyze ATP, ADP, AMP, PPi, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, and β-glycerophosphate; however, only a few compounds have been considered as natural in vivo AP substrates, like PPi, pyridoxal-5′-phosphate (PLP or vitamin B5), and phosphoethanolamine (PEA). APs by themselves are extremely efficient ATPase enzymes, but an autoregulatory mechanism has been described in order to modify the substrate specificity depending on the environmental concentrations of free inorganic phosphates or cell and tissue demands. Pi itself is known to inhibit the hydrolytic activity through a competitive mechanism, and therefore Pi levels will impact the ability of AP to hydrolyze PPi [15, 16]. Levamisole is an inhibitor of the APs. Because of the ability to cleave all forms of adenosine phosphates, APs

The protocol detailed here for the detection of E-NTPDases, E-NPPs, and eN is based on the Wachstein-Meisel lead phosphate method [18], and the protocol for AP identification is based on the Gossrau method [19], with some modifications.

Tissue pieces are fixed with 4% paraformaldehyde for a time period varying from a few hours to 2 days depending on the size (**Figure 2**). Following fixation, tissue pieces are cryopreserved by embedding them in 30% sucrose (in Milli-Q H2O) O/N or until tissue sinks. Tissues are then embedded in optimum cutting temperature (OCT) compound and cut with a cryostat into 15 μm-thick sections that are put on slides. It is recommended that pretreated slides be used, either homemade polylysine-treated or the commercial ones, to eliminate tissue loss during the

Tissue slides are rinsed with phosphate buffered saline (PBS) to remove OCT compound and washed twice with 50 mM Tris-maleate buffer pH 7.4 at RT. Slides are then incubated for 30 min at RT with preincubation buffer (50 mM Tris-maleate buffer pH 7.4 containing 2 mM MgCl2 and 250 mM sucrose) and then for 1 h at 37°C with the enzyme reaction buffer (50 mM Tris-maleate buffer pH 7.4 supplemented with 250 mM sucrose, 2 mM MgCl2, 5 mM MnCl2, 2 mM Pb(NO3)2, and 2 mM

**118**

*Scheme including the main steps of the lead phosphate-based method for ecto-nucleotidase activity detection.*

CaCl2 and stabilized with 3% Dextran T-250) in the presence or absence of nucleotide as substrate (e.g., ATP). The incubation time and the substrate concentration may vary depending on the experiment, but 1 h at 1 mM is generally suitable. To avoid interference with AP activity, experiments are performed in the presence of the inhibitor levamisole (2.5 mM). Note that the optimum pH for APs is 9, but they are also active at a pH of 7.4. The reaction is stopped with dH2O and revealed by incubating with 1% (NH4)2S v/v for exactly 1 min. A control slide in the absence of nucleotide, in which no reaction is expected, is included in the experiment. Nuclei are counterstained with hematoxylin. Samples are then mounted with aqueous mounting medium (e.g., Fluoromount™, Sigma-Aldrich); dehydration is not recommended because of the eventual loss of lead precipitates. Finally, samples are observed and photographed under light microscope; enzyme-active sites are brownish black. An adapted protocol with slight modifications, including the replacement of ammonium sulfide by glutaraldehyde, can be applied for electron microscopy [20]. **Table 1** includes buffer formulations.



#### **Table 1.**

*Formulation of buffers used for the lead phosphate-based method. The most right column includes the reagent amounts calculated to prepare a 1 mL final volume (FV) solution. Different nucleotides can be used as substrate. Inhibitors can also be added to both preincubation and reaction buffers; H2O to adjust the volume must then be modified accordingly.*

Besides levamisole, enzyme inhibitors might be included in both preincubation and incubation buffers. For example, 1 mM α,β-methylene-ADP efficiently inhibits CD73, and POM 1 inhibits NTPDases (**Figure 3**).

**121**

*2.1.2 In cell cultures*

**Figure 3.**

reported for the tissue slices.

**2.2 Gossrau-based method for APs**

*In Situ Identification of Ectoenzymes Involved in the Hydrolysis of Extracellular Nucleotides*

Cells are seeded onto coverslips and allowed to grow with their regular medium until the desired confluence is achieved. The medium is then removed, and the cells are washed twice with PBS before fixation with 4% paraformaldehyde for 5–10 min at RT. Cells are washed three times with PBS to wash out the fixative and kept at 4°C with PBS until use. To proceed with the protocol, coverslips are washed twice for 5 min with gentle rocking with 50 mM Tris-maleate buffer pH 7.4 at RT and then incubated for 30 min at RT with the preincubation buffer. The following steps are as

*(A) In situ AMPase activity in the endometrial carcinoma human HEC-1B cell line in the presence of 1 mM AMP (a). Note that most of the activity is located at the cell membrane, where CD73 is expressed. Incubation in the presence of the inhibitor α,β-methylene-ADP (α,β-meADP) drastically diminished the activity (b). No AMPase activity is detected when AMP is omitted in the reaction (c). (B) In situ enzyme ATPase activity in human endometrium in the presence of 1 mM ATP (a). Activity is strongly inhibited with the NTPDase inhibitor POM 1 (b). No ATP activity is detected when ATP is omitted (c). Scale bars are 50 μm (a) and 200 μm (B).*

In situ localization of APs can be addressed by using the Gossrau method that utilizes nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as artificial substrates for the APs. Briefly, tissue slices or fixed cells grown on coverslips are washed twice in 0.1 M Tris–HCl buffer pH 7.4 containing 5 mM MgCl2 and then preincubated with the same buffer at pH 9.4 for 15 min at RT. Enzymatic reaction starts by adding the revealing reagent BCIP/NBT (Sigma-Aldrich) for 7 min (up to 15 min) at RT and stopped with 0.1 M Tris–HCl buffer pH 7.4. In AP inhibition experiments, 5 mM levamisole can be added to both preincubation and enzyme reaction buffers. In control experiments, the revealing reagent is omitted. Since the reaction generates blue precipitates, nuclei staining with hematoxylin should be avoided. Alternatively nuclei can be counterstained with methyl green dye. Samples are then mounted with aqueous mounting medium (e.g., Fluoromount™, Sigma-

Aldrich) and observed and photographed under light microscope.

*DOI: http://dx.doi.org/10.5772/intechopen.84495*

*In Situ Identification of Ectoenzymes Involved in the Hydrolysis of Extracellular Nucleotides DOI: http://dx.doi.org/10.5772/intechopen.84495*

#### **Figure 3.**

*Immunohistochemistry - The Ageless Biotechnology*

microscopy [20]. **Table 1** includes buffer formulations.

CaCl2 and stabilized with 3% Dextran T-250) in the presence or absence of nucleotide as substrate (e.g., ATP). The incubation time and the substrate concentration may vary depending on the experiment, but 1 h at 1 mM is generally suitable. To avoid interference with AP activity, experiments are performed in the presence of the inhibitor levamisole (2.5 mM). Note that the optimum pH for APs is 9, but they are also active at a pH of 7.4. The reaction is stopped with dH2O and revealed by incubating with 1% (NH4)2S v/v for exactly 1 min. A control slide in the absence of nucleotide, in which no reaction is expected, is included in the experiment. Nuclei are counterstained with hematoxylin. Samples are then mounted with aqueous mounting medium (e.g., Fluoromount™, Sigma-Aldrich); dehydration is not recommended because of the eventual loss of lead precipitates. Finally, samples are observed and photographed under light microscope; enzyme-active sites are brownish black. An adapted protocol with slight modifications, including the replacement of ammonium sulfide by glutaraldehyde, can be applied for electron

Besides levamisole, enzyme inhibitors might be included in both preincubation and incubation buffers. For example, 1 mM α,β-methylene-ADP efficiently inhibits

*Formulation of buffers used for the lead phosphate-based method. The most right column includes the reagent amounts calculated to prepare a 1 mL final volume (FV) solution. Different nucleotides can be used as substrate. Inhibitors can also be added to both preincubation and reaction buffers; H2O to adjust the volume* 

CD73, and POM 1 inhibits NTPDases (**Figure 3**).

**120**

**Table 1.**

*must then be modified accordingly.*

*(A) In situ AMPase activity in the endometrial carcinoma human HEC-1B cell line in the presence of 1 mM AMP (a). Note that most of the activity is located at the cell membrane, where CD73 is expressed. Incubation in the presence of the inhibitor α,β-methylene-ADP (α,β-meADP) drastically diminished the activity (b). No AMPase activity is detected when AMP is omitted in the reaction (c). (B) In situ enzyme ATPase activity in human endometrium in the presence of 1 mM ATP (a). Activity is strongly inhibited with the NTPDase inhibitor POM 1 (b). No ATP activity is detected when ATP is omitted (c). Scale bars are 50 μm (a) and 200 μm (B).*

#### *2.1.2 In cell cultures*

Cells are seeded onto coverslips and allowed to grow with their regular medium until the desired confluence is achieved. The medium is then removed, and the cells are washed twice with PBS before fixation with 4% paraformaldehyde for 5–10 min at RT. Cells are washed three times with PBS to wash out the fixative and kept at 4°C with PBS until use. To proceed with the protocol, coverslips are washed twice for 5 min with gentle rocking with 50 mM Tris-maleate buffer pH 7.4 at RT and then incubated for 30 min at RT with the preincubation buffer. The following steps are as reported for the tissue slices.

#### **2.2 Gossrau-based method for APs**

In situ localization of APs can be addressed by using the Gossrau method that utilizes nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as artificial substrates for the APs. Briefly, tissue slices or fixed cells grown on coverslips are washed twice in 0.1 M Tris–HCl buffer pH 7.4 containing 5 mM MgCl2 and then preincubated with the same buffer at pH 9.4 for 15 min at RT. Enzymatic reaction starts by adding the revealing reagent BCIP/NBT (Sigma-Aldrich) for 7 min (up to 15 min) at RT and stopped with 0.1 M Tris–HCl buffer pH 7.4. In AP inhibition experiments, 5 mM levamisole can be added to both preincubation and enzyme reaction buffers. In control experiments, the revealing reagent is omitted. Since the reaction generates blue precipitates, nuclei staining with hematoxylin should be avoided. Alternatively nuclei can be counterstained with methyl green dye. Samples are then mounted with aqueous mounting medium (e.g., Fluoromount™, Sigma-Aldrich) and observed and photographed under light microscope.

#### **Figure 4.**

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

**123**

**Figure 5.**

*in situ enzyme activity, Villamonte et al. [21].*

*In Situ Identification of Ectoenzymes Involved in the Hydrolysis of Extracellular Nucleotides*

**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 visualiza-

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

**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

*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* 

hampering of fluorescence capture by the dark brown lead deposits.

*DOI: http://dx.doi.org/10.5772/intechopen.84495*

tion) are taken sequentially from the same field.

*In Situ Identification of Ectoenzymes Involved in the Hydrolysis of Extracellular Nucleotides DOI: http://dx.doi.org/10.5772/intechopen.84495*
