The work presented in this study was financially supported by the Slovenian Research Agency (research core funding nos. P3-0396 and I0-0029, as well as research projects nos. J3-9289, N3-0048, and N3-0133) and by the Austrian Science Fund / Fonds zur F&#246;rderung der Wissenschaftlichen Forschung (bilateral grants I3562--B27 and I4319--B30). We thank Maru&#353;a Ro&#353;er, Ma&#353;a &#268;ater, and Rudi Mlakar for excellent technical assistance.

The authors declare that the research was conducted in the absence of any commercial or financial interest.

The pancreatic tissue slice method is a fast experimental method to study the morphology and physiology of the endocrine and exocrine parts of the pancreas in a more conserved, in situ preparation. Many of the advantages have already been pointed out in the Introduction. It is worth pointing out that in general (i.e., not only for calcium imaging), the slice approach to study pancreatic physiology saves time because it does not involve a recovery period after isolation. The latter is not absolutely necessary with all types of experiments and uses of isolated islets from different species, but is typically employed to increase purity, restore viability and functionality, and sometimes to collect islets from several donors59,60,61,62,63,64. However, in the context of calcium imaging, beta cell responses have been found to depend on culture duration and conditions, and this is an important source of variation that should be taken into account when using isolated islets15,65. The same issue should be considered for tissue slices if their long-term culture becomes a widely used option in the future22,36. The tissue slice method also has a high yield and thus potentially reduces animal suffering and increases statistical power. Moreover, as many slices can be prepared from a single animal and because the slices survive for long periods, including the same animal or even the same islet in both the experimental and the control groups becomes feasible.
As the original architecture and cell-to-cell communications are preserved, and because it is compatible with a number of structural analyses, electrophysiological, imaging methods, and hormone secretion assays, this method is especially useful to study pancreatic functions that depend on undisturbed interactions between individual cells, e.g., sensitivity to secretagogues, paracrine and immune interactions between different cell types, patterns of electrical activity, properties of calcium dynamics, and the secretion of different hormones. For calcium imaging specifically, the main advantages of using slices are the exposure of the islet core and the possibility to acquire signals from many different cell types with high resolution. Depending on the requirements of the experiment and the age of animals, the thickness can be varied, the slices can be transfected, or obtained from animals with genetically encoded reporters. As explained in more detail below, the latter two approaches also enable specific functional identification and characterization of responses from non-beta cells 31,66. Moreover, islets from well-defined parts of the organ can be studied for differences in responsiveness or susceptibility to disease. Although they do not require a recovery incubation period, they can easily be incubated with different pharmacological agents, fatty acids, high glucose, and cytokines.
Most importantly, as high resolution is achievable in combination with single-cell or even subcellular resolution, confocal calcium imaging in slices is one of the most suitable methods for analyzing calcium waves, functional connectivity, and the different functional roles of cells in distinct parts of an islet54,67. Despite a number of advantages, the tissue slice approach has important limitations. First, it is still at least partly disruptive to islet and exocrine architecture, especially at the cut surface, and precautions, such as low temperature, frequent exchange of solutions, and gentle and quick manipulation, are needed during preparation to prevent additional mechanical and endogenous enzymatic damage. Second, the patterns of nutrient and secretagogue delivery are still inferior to the in vivo route, the preparation is detached from systemic innervation, and inter-organ feedback, such as between the islet and its target tissues, is impossible, in contrast to in vivo approaches. Third, maximum slice thickness is limited by oxygenation, nutrient delivery, and pH regulation at ~200 &#181;m9. Further, both preparation of slices and imaging need a lot of training, and in-depth analyses of calcium data from long time series and from many cells require specialized knowledge that is often not included in the toolkit of a classical physiologist and requires help from physicists or data scientists. The advantage that homo- and heterotypic interactions are preserved can also complicate the analysis of samples due to the presence of signals from other cells in regions of interest. Depending on protocols, activation of other cells can lead to indirect additional stimulation or inhibition of an observed cell.
This can only be resolved conclusively by deconvolution approaches, by more complex stimulation protocols including substances that block some of the indirect effects, by using specific knock-out animals, and by careful comparison of results with results from other studies employing more reductionist methodologies. Additionally, if secretion measurements are necessary, it should be kept in mind that some slices may lack islets, and the total mass of endocrine tissue in a single slice is typically low. The preparation of acute pancreatic tissue slices for imaging involves several critical steps discussed in the following sections and summarized in Table 1, where the reader can also find short, but important tips for troubleshooting. First, when preparing the agarose solution, the agarose powder must dissolve completely, otherwise the undissolved particles may obstruct the injection. Keep the homogeneous agarose solution at 37-45 &#176;C to prevent hardening of agarose due to too low a temperature on the one hand and to prevent tissue damage due to too high temperatures on the other hand. After use, the remaining agarose may be stored at 4 &#176;C and reheated, although repeated reheating can result in increased density due to water evaporation, eventually making the injection difficult or impossible.
The next critical step in the preparation is clamping the major duodenal papilla correctly. A white spot on the duodenum indicates the junction of the common bile duct and the duodenum. A clamp placed too proximally will result in obstruction of some lateral pancreatic branches of the common duct, disabling the injection of these parts, whereas a clamp placed too distally will result in agarose leaking through the lower resistance path directly into the duodenum. Before cannulation of the common bile duct, the surrounding adipose tissue can be carefully removed for better visualization of the duct and greater control during injection. Insufficient precision during removal of the surrounding tissue may result in perforation of the duct. The selection of the needle diameter used for agarose injection is also important. In mice, a 30 G needle is preferably used; smaller (32 or 33 G) needles require more effort due to high viscosity of the agarose solution and are more prone to obstruction. However, if used in combination with a lower-density agarose solution, they can be very helpful in smaller mouse strains and younger animals. During the initial postnatal days, agarose may alternatively be injected subcapsularly rather than intraductally2. Using needles with greater diameter in mice will most probably result in damaging the common bile duct. This can also happen with the correct needle diameter, and a forceps can help in keeping the needle in place during injection. Larger diameter needles may be the only solution in case of larger ducts, as found in rats. If the needle is too narrow to ensure a tight seal preventing back-leakage, a ligature may be placed around it upon successful entry into the duct.
Agarose injection takes some effort due to the solution&#39;s viscosity, and once the injection process has started, it should not be interrupted as the low-melting-point agarose solution may solidify in the needle or the largest parts of the ductal tree before the injection is completed. This will result in poor tissue penetration and worse support during cutting. The duct should always be cannulated at the point where the left hepatic duct and the cystic duct join to form the common bile duct.. If the common bile duct gets perforated, repeatedly try cannulating closer to the duodenum. When the pancreas is sufficiently stabilized with agarose solution and extracted from the peritoneal cavity, small pieces of well-injected tissue are cut. Before embedding them into the agarose, it is crucial to remove all the adipose and connective tissues as their residues make slicing more challenging. The same applies to blood vessels and duct residues, except when they are the focus of the experiment. In this case, make sure to position them in such a way that the desired cross-section will be obtained. When embedding the tissue in agarose, ensure that the temperature is appropriate (37 &#176;C), and that the tissue is completely surrounded by agarose, as forces during vibratome slicing can rip out the pancreas tissue from the agarose blocks.
Quickly drying the tissue blocks before placing them in agarose by placing them briefly on a paper tissue can help prevent poor contact between tissue and agarose during this step. During solidification of agarose blocks, place the Petri dish horizontally, and prevent contact between the pancreas tissue and the bottom of the Petri dish. If the pancreas is not fully injected, the cutting process will be challenging. Therefore, try to reduce the cutting speed to obtain tissue slices. To minimize cell damage during vibratome slicing, replace the ECS (and the ice cubes made of ECS) in the slicing chamber regularly. The latter will reduce the activity of pancreatic enzymes released from acinar tissue during slicing. The thickness of the slices is also of crucial importance. For calcium dynamics and electrophysiological experiments, 140 &#181;m slices are usually cut; however, according to the aim of the study, slice thickness can range from 90 &#181;m to 200 &#181;m. Keep in mind that in thicker slices, the diffusion of oxygen and nutrients will be limited, but they will include more tissue. Additionally, the proportion of uncut islets may be expected to increase with increasing slice thickness. Slices can be stored in a regularly exchanged ECS at room temperature for several hours or even cultivated in an appropriate cell medium for several days; however, this may eventually affect the normal islet cell physiology3,22.
When preparing the dye solution, ensure careful mixing of all components, and avoid exposure to ambient light. The pancreatic slice is composed of many cell layers, and the uptake of calcium dye is limited to the first few most superficial cell layers, as described previously for isolated islets58,68, and pituitary slices69. However, in contrast to isolated islets where the surrounding capsule and outer cell layers hinder the penetration of the dye into deeper layers, tissue slices permit access to the entire cross-sectional surface of the islet, enabling simultaneous measurement of calcium dynamics in hundreds of cells from all layers of an islet. Fluorescent Ca2+ indicators are the most widely used for measuring calcium dynamics, and together with CLSM, they enable recordings with high temporal resolution, reaching several hundred Hertz. When selecting the most appropriate fluorescent Ca2+ indicator, consider different factors, including the indicator form, which influences the cell loading method, measurement mode (qualitative or quantitative), and dissociation constant (Kd) that needs to be in the Ca2+ concentration range of interest and depends on pH, temperature, presence of Mg2+ and other ions, as well as protein binding. As cellular Ca2+ signals are usually transient, the Ca2+ binding rate constant should also be considered. For measuring [Ca2+]IC dynamics in pancreatic cells, this group mainly uses the cell-permeable Ca2+ indicator dye described in this protocol (Table of Materials) as it is a long wavelength indicator with the emission wavelengths in the spectrum where cellular autofluorescence is usually less problematic, and the energy of excitation light is low, which reduces the potential for cellular photodamage. Because this dye is fluorescent at low Ca2+ concentrations, this facilitates the determination of baseline [Ca2+]IC and increases cellular visibility before stimulation. After binding Ca2+, the fluorescence intensity of the dye increases 14-fold, enabling detection of even slight changes in [Ca2+]IC.
For successful live-cell calcium imaging, several crucial hardware parameters need to be considered, as described in the protocol section. For live-cell imaging wherein signal amplitudes are low and chances of phototoxicity are high, objectives with a higher NA are preferably used to collect more light from the specimen. If calcium dynamics must be recorded with a high temporal resolution, use the resonant scanner instead of linear galvanometers. Besides choosing the right objective, the use of highly sensitive detectors-such as hybrid detectors that require less laser power-avoids phototoxicity and photobleaching. This is of special importance for long-lasting calcium imaging. Other important steps in calcium imaging are parameter settings of image quality for time series acquisitions. The most important are the temporal and the spatial resolution. As the calcium dynamics per se determines the lowest acceptable temporal resolution, the sampling rate needs to be at least two-fold higher than the expected signal frequency to detect the signal or even 10 times higher to detect the shape of the signal reliably. In acute pancreatic tissue slices, calcium dynamics can be measured in hundreds of cells simultaneously and therefore, the spatial resolution is also important. This can be enhanced by increasing the number of pixels or by increasing the line averaging during live acquisition. However, because of the inverse relationship between the spatial and the temporal resolution, a trade-off between both settings is needed.
If calcium imaging has to be performed in a specific cell population within the pancreas, a stimulus able to functionally differentiate the cells within the slice is necessary. High glucose reliably and quickly activates beta cells to an oscillatory pattern that is superimposed on an elevated calcium level and is highly synchronized among all cells within an islet32,58,70. The beta cells are the most numerous cell type within an islet and are located mostly in the islet core in mice. The same stimulation protocol decreases and sometimes does not appreciably change the bursting in alpha cells30,32,58,70,71,72. To discriminate alpha cells functionally, low (3 mM) glucose, glutamate or adrenalin can be used to increase their frequency or basal [Ca2+]IC21,72,73,74,75. They represent 10-20% of islet cells and will be detected on the islet periphery1. Delta cells are also found on the periphery. They make up only ~5% of the total number of endocrine cells in an islet and are typically active in 6 mM glucose and respond to glucose stimulation with an increased irregular bursting activity from the baseline or a slightly elevated calcium level1,32,71,76. Ghrelin can be used for specific stimulation of delta cells21,77,78,79 in calcium imaging experiments. However, protocols for specific functional identification of PP and epsilon cells remain to be defined. Further, 25 nM acetylcholine reliably activates acinar cells into bursting activity35,80,81. Additionally, a number of other secretagogues, such as cerulein, cholecystokinin, and carbamylcholine, can be used to evoke calcium responses in acinar cells22,40,82,83.
Finally, 1 mM chenodeoxycholic acid reliably evokes calcium responses in ductal cells in tissue slices; angiotensin II, ATP, and some other secretagogues can also be used11,23,84,85. Whenever a functional identification based on characteristic responses to specific secretagogues and inhibitors is not sufficient, genetically labelled animals31, transfected cells73, or immunocytochemistry can be employed for the identification of different cell types9,22,71,86. During the last couple of years, the tissue slice method has been successfully adapted to human tissue, opening many new important research avenues in both exocrine41 and endocrine physiology9,36,37,39. Interestingly, a detailed assessment of calcium dynamics in human islets has been notoriously difficult and remains to be investigated in greater detail87. Combined with advanced confocal microscopy, the pancreatic tissue slice method has enabled many new insights into the calcium dynamics in mice and will hopefully do the same for human tissue.

The mammalian pancreas is a large exocrine and endocrine gland. The exocrine part makes up 96-99% of the total pancreas volume and consists of acini and ducts. The endocrine part is made up of a large number of islets of Langerhans accounting for the remaining 1-4% of the total pancreas volume1. The exocrine part secretes major digestive enzymes that break down energy-rich polymers in food, as well as a bicarbonate-rich fluid, which combines with other gastrointestinal secretions to provide an environment suitable for the action of enzymes. The endocrine part secretes hormones that regulate postprandial distribution, storage, and interprandial release of energy-rich nutrients. Although the exocrine tissue is relatively underdeveloped and the endocrine relatively well-developed at birth, the former quickly overgrows the latter upon weaning2,3,4. Early studies of pancreatic function marked the birth of modern physiology, and major methodological advancements in the field have been followed by major scientific breaktroughs5. Working with the pancreas is technically challenging due to the intricate structure of the gland, but is also a big motivation because of diseases such as pancreatic cancer, pancreatitis, and diabetes that present major threats to public health, and for which novel therapeutic approaches are needed.
Isolated islets6, acini7,8, and ductal fragments had been developed and used for decades as gold standard methods owing to their advantages compared with cell lines and primary dispersed endocrine, acinar, and ductal cells9,10. Despite the markedly improved function of isolated cell collectives, these methods still involve considerable mechanical and enzymatic stress, isolate cells from the surrounding tissue and thus lack paracrine interactions and mechanical support, and most importantly, are accompanied by significant changes to normal physiology11,12,13. The acute mouse pancreatic tissue slice was developed in 2001 out of a perceived need to develop an experimental platform similar to brain, pituitary, and adrenal slices with preserved intercellular contacts, paracrine interactions, mesenchyme, and tissue architecture, as well as without some of the most important shortcomings of the gold standard method in islet research of that time-the isolated islets12,14. Among these shortcomings are damage to the outermost layers, lack of accessibility of core islet areas, and the need for cultivation with possibly important effects on cell identity and physiology12,15. Moreover, the tissue slice method enables studies on animal models with grossly deranged islet architecture where it is impossible to isolate islets, or when islet yield is extremely low by traditional isolation16,17,18,19,20,21.
Additionally, the slice is more suitable for studying morphological changes during the development of diabetes and pancreatitis, for instance, as it enables a better overview of the whole tissue and is also compatible with studying regional differences. Importantly, despite the early focus on the endocrine part, the tissue slice method inherently enables the study of the exocrine components9,22,23. During the first decade after its introduction, the method was employed for electrophysiological studies of beta14,24,25,26,27,28,29 and alpha30,31 cells as well as for examining the morphological and functional maturation of the pancreas2,3. A decade later in 2013, the method was successfully adapted for live-cell calcium imaging of islet cells using CLSM to characterize their responses to glucose32, their functional connectivity patterns33, and the relationship between membrane potential and intracellular calcium by combining a fluorescent calcium dye with a membrane potential dye34. Later in the same year, the method was also used to assess calcium&#160;dynamics in acinar cells22,35. Over the following years, pancreatic tissue slices have been used in a number of different studies and successfully adapted to pig and human tissue9,36,37,38,39,40,41. However, taken together, calcium imaging-in mouse pancreatic tissue slices in general and in islets in particular-is still mostly performed by this group. One of the main reasons for this may lie in the combination of a technically challenging tissue slice preparation, the need for a confocal microscope, and rather complex data analysis. The main aim of the present paper is to make this powerful method more accessible to other potential users.
There are already some excellent methodological articles dealing in detail with tissue slice preparation and the use of slices for structural and secretion studies, but not for confocal calcium imaging9,42,43. Therefore, this paper focuses on some additional tips and tricks during the preparation of slices, on steps critical for successful dye loading, image acquisition, as well as on the main steps of basic calcium data analysis. Therefore, this contribution should be viewed as being complementary to rather than as an alternative for the abovementioned method. Similarly, calcium imaging in mouse pancreatic tissue slices shall be viewed as an experimental approach to be used to answer specific questions and is thus complementary to rather than an absolute alternative for other calcium imaging approaches in pancreatic physiology such as isolated ducts or acini, isolated islets, organoids, islets transplanted into the anterior chamber of the eye, and recordings in vivo11,44,45,46,47,48. The promise of calcium imaging in mouse pancreatic tissue slices is probably best illustrated by recent successful recordings of calcium dynamics in islet mesenchymal cells such as pericytes49 and macrophages50, as well as in ductal cells23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance KERN ALJ 120-4</td>
			<td>KERN &#38; SOHN GmbH</td>
			<td>ALJ 160-4A</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope Leica TCS SP5 II Upright setup</td>
			<td>Leica</td>
			<td>5100001578</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope Leica TCS SP5 AOBS Tandem II setup</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cork pad 15 cm x 15 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;15 mL centrifuge tubes</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>CLS430790</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;Round Ice Bucket with Lid, 4 L</td>
			<td>Fischer Scientific, Leicestershire, UK</td>
			<td>432124</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge razor blade</td>
			<td>Personna, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps</td>
			<td>FST, Germany</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 0.5 mL</td>
			<td>Eppendorf</td>
			<td>0030 121.023</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask 200 mL</td>
			<td>IsoLab, Germany</td>
			<td>027.01.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - ToughCut</td>
			<td>FST, Germany</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat orbital shaker&#160; IKA KS 260 basic</td>
			<td>IKA</td>
			<td>Ident. No.: 0002980200</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass lab bottle 1000 mL</td>
			<td>IsoLab, Germany</td>
			<td>091.01.901</td>
			<td></td>
		</tr>
		<tr>
			<td>Hartman Hemostat, curved</td>
			<td>FST, Germany</td>
			<td>13003-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HCX APO L 20x/1.00 W HCX APO L (water immersion objective, 20x, NA 1.0)</td>
			<td>Leica</td>
			<td>15507701</td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring cylinder 25 mL</td>
			<td>IsoLab, Germany</td>
			<td>015.01.025</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator Control box SM-7, Keypad SM-7</td>
			<td>Luigs &#38; Neumann</td>
			<td>&#160;200-100 900 7311, 200-100 900 9050</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave owen</td>
			<td>Gorenje, Slovenia</td>
			<td>MO20MW</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmometer Gonotec 010</td>
			<td>Gonotec, Berlin, Germany</td>
			<td>OSMOMAT 010 Nr. 01-02-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td>Faber-Castell, No.2</td>
			<td></td>
			<td>Any thin soft round paint brush No.2, preferably black</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perifusion pumps</td>
			<td>Ismatec</td>
			<td>ISM 827</td>
			<td>Reglo Analog MS - 4/8</td>
		</tr>
		<tr>
			<td>Petri dish 100/20 mm</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35/10 mm</td>
			<td>Greiner bio-one</td>
			<td>627102</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35 x 10 mm Nunclon Delta</td>
			<td>Thermo Fischer Scientific, Waltham, MA USA</td>
			<td>153066</td>
			<td>NON-STICKY for agarose blocks</td>
		</tr>
		<tr>
			<td>pH meter inoLab pH Level 1</td>
			<td>WTW, Weilheim, Germany</td>
			<td>E163694</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 1000 mL</td>
			<td>Eppendorf</td>
			<td>3121 000.120</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 50 mL</td>
			<td>Eppendorf</td>
			<td>3121 000.066</td>
			<td></td>
		</tr>
		<tr>
			<td>Push pins 23 mm</td>
			<td>Deli, Ningbo, China</td>
			<td>E0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw cap tube, 15 mL</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>Semken Forceps</td>
			<td>FST, Germany</td>
			<td>11008-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilizing ring for Erlenmeyer flask</td>
			<td>IsoLab, Germany</td>
			<td>027.11.048</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope Nikon SMZ 745</td>
			<td>Nikon, Melville, NY USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Injekt Solo 5 mL</td>
			<td>Braun, Melsungen, Germany</td>
			<td>4606051V</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe needle 0.30 x 12 mm (30 G x 1/2&#34;)</td>
			<td>Braun, Melsungen, Germany</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Luigs &#38; Neumann</td>
			<td>200-100 500 0150, 200-150-500-145</td>
			<td>Slice mini chamber, Temperature controller TC 07</td>
		</tr>
		<tr>
			<td>Tubings for perifusion system</td>
			<td>Ismatec</td>
			<td>SC0310</td>
			<td>Ismatec Pharmed 1.14 mm(ID) + silicone tubing 1.0 (ID) x 1.8 mm(OD)</td>
		</tr>
		<tr>
			<td>Ultrasonic bath Studio GT-7810A</td>
			<td>Globaltronics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome Leica VT 1000 S</td>
			<td>Leica, Nussloch, Germany</td>
			<td>14047235613</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric flask 1000 mL</td>
			<td>IsoLab, Germany</td>
			<td>013.01.910</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer Neolab 7-2020</td>
			<td>Neolab</td>
			<td>&#160;7-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Thermo Haake open-bath circulator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Z527912</td>
			<td></td>
		</tr>
		<tr>
			<td>Material/Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate - CaCl2.2H2O</td>
			<td>Sigma Aldrich, Germany</td>
			<td>C5080-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma Aldrich, Germany</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D4540-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-lactic acid</td>
			<td>Sigma Aldrich, Germany</td>
			<td>L1250-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Merck KGaA, Darmstadt, Germany</td>
			<td>D8662-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas mixture containing 95% O2 and 5% CO2 at barometric pressure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glue Wekem sekundenkleber WK-110</td>
			<td>Wekem GmbH, Bergkamen, Germany</td>
			<td>WK 110-020</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich, Germany</td>
			<td>H3375-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-(+)-ascorbic acid</td>
			<td>Sigma Aldrich, Germany</td>
			<td>A9,290-2</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td>Thermo Fischer Scientific, Waltham, MA USA</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate - MgCl2.2H2O</td>
			<td>Sigma Aldrich, Germany</td>
			<td>M2670-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Myo-inositol</td>
			<td>Sigma Aldrich, Germany</td>
			<td>I5125-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Oregon Green 488 BAPTA-1, AM</td>
			<td>Invitrogen (Thermo FisherScientific)</td>
			<td>O6807</td>
			<td>cell-permeable Ca2+ indicator (excitation/emission: 495/523 nm)</td>
		</tr>
		<tr>
			<td>Pluronic F-127 (20% Solution in DMSO)</td>
			<td>Invitrogen (Thermo Fisher Scientific)</td>
			<td>P3000MP</td>
			<td>polaxamer: nonionic triblock copolymer</td>
		</tr>
		<tr>
			<td>Potassium chloride - KCl</td>
			<td>Sigma Aldrich, Germany</td>
			<td>31248</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaPlaque GTG agarose</td>
			<td>Lonza, Rockland, USA</td>
			<td>50111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate - NaHCO3</td>
			<td>Honeywell, Germany</td>
			<td>31437-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride - NaCl</td>
			<td>Honeywell, Germany</td>
			<td>31434-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide - NaOH</td>
			<td>Sigma Aldrich, Germany</td>
			<td>30620</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic- NaH2PO4</td>
			<td>Sigma Aldrich, Germany</td>
			<td>S0751-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich, Germany</td>
			<td>15990-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td>FIJI is an open source project</td>
		</tr>
		<tr>
			<td>LASAF</td>
			<td>Leica microsystems, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Python</td>
			<td>Python Software Foundation</td>
			<td></td>
			<td>Python is an open source project</td>
		</tr>
	</tbody>
</table>

confocal laser scanning microscopy of calcium dynamics in acute mouse pancreatic tissue slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

The injection of the agarose solution into the pancreatic duct is the most critical step in pancreas tissue slice preparation. A successful injection can be recognized by a whitening of the pancreas tissue, as seen on the left side of Figure 3A, while an incompletely injected part of the pancreas is presented on the right side of Figure 3A. The islets of Langerhans can be recognized by the naked eye or under a stereomicroscope, and this aids in cutting the appropriate parts of the pancreas for subsequent embedding in agarose blocks (Figure 3B). In a freshly cut mouse pancreatic tissue slice, islets of Langerhans can be easily distinguished from the surrounding exocrine tissue and mesenchyme as white spots under the stereomicroscope (Figure 3C) or as brownish structures under the light microscope (Figure 3D). The pancreatic tissue slices can be used for distinct types of experiments for at least 12 h after slicing. In addition to the gross morphological assessment under the stereomicroscope, the light microscope, and the functional responses of cells during calcium imaging, the viability of the pancreatic tissue slices can be assessed (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62293/62293fig04.jpg" /> 
Figure 4: Viability of cells within the tissue slice. Viability of cells was determined with the Live/Dead assay. Live cells are stained by Calcein AM (shown in green), while dead cells are stained with ethidium homodimer-1 (shown in red). Yellow lines denote the position of the X-Y cross section of the Z-stack displayed at the bottom and the right. The full depth of the Z-stack is 88 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62293/62293fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For calcium imaging experiments, the fluorescent calcium indicator needs to penetrate through a few layers of cells. Figure 5A presents successful loading of the cell-permeable Ca2+ indicator dye into the pancreatic tissue slice in which individual islet and acinar cells can be recognized. In contrast, slices in Figure 5B-D are not optimal due to unsuccessful penetration of the dye (Figure 5B), lack of islet cells (Figure 5C), and a lot of necrotic tissue on the surface (Figure 5D). Such slices can be discarded, checked for the presence of additional islets that are cut or stained better (see Table 1 for troubleshooting), or used for recording the responses of exocrine cells.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62293/62293fig05.jpg" /> 
Figure 5: Examples of usable and unusable preparations. (A) An example of a successful preparation of the pancreas tissue slice with well-stained cells in the islets of Langerhans, as well as ductal cells and surrounding acinar tissue. (B) An example of a poorly stained tissue slice. (C) Example of an islet of Langerhans with structural discontinuations. (D) An example of an islet of Langerhans containing many dead cells and a lot of debris. The &#34;glow-over, glow-under&#34; lookup table on the right displays 0 intensity in green and saturation in blue. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62293/62293fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Representative results from calcium imaging using the cell-permeable Ca2+ indicator dye are shown in Figure 6. In Figure 6A, a high-resolution image of a pancreatic tissue slice is presented, containing an islet of Langerhans, acinar tissue, and a pancreatic duct. For better distinction, the endocrine, exocrine, and ductal part of the pancreatic tissue slice presented in Figure 6A are colored in Figure 6B. Using appropriate stimuli can functionally discriminate between different islet cells, or islet and non-islet cells51. Beta cells will typically respond to a square-pulse stimulation by glucose with a transient increase in [Ca2+]IC followed by fast calcium oscillations on a sustained plateau (Figure 6C, upper panel).
As all beta cells are coupled into a single, large, functional syncytium, these oscillations are also very well synchronized among different cells by means of spreading [Ca2+]IC waves32,34,52,53,54 (Figure 7C). Slower [Ca2+]IC oscillations with a period of 5-15 minutes may underlie the fast oscillations or even be the predominant type of reponse55,56. The same simple protocol may reveal other types of responses, especially at the periphery of islets (Figure 6C, lower panel). As these cells are not synchronized with beta cells and respond with faster and more irregular oscillations that are already present in low glucose conditions or with a decrease in activity, such responses are highly suggestive of non-beta cells21,32,57,58. However, their definitive functional characterization requires more complex protocols with additional stimulation steps or alternative approaches, which are discussed below. Typical responses of acinar and ductal cells are presented in Figure 6D and Figure 6E, respectively. Refer to the literature for more details on acinar and ductal cells22,23,35.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62293/62293fig06.jpg" /> 
Figure 6: Representative results of calcium dynamics in distinct types of pancreatic cells. (A) A high-resolution image of an islet of Langerhans with surrounding tissue. Scale bar = 100 &#181;m. (B) Delineation of distinct parts of pancreatic tissue with acinar tissue shown in yellow, an islet of Langerhans shown in red, and a segment of the ductal tree in blue. Scale bar = 100 &#181;m. (C) Typical traces of calcium dynamics in beta and putative non-beta cells during stimulation with 12 mM glucose; 3 mM glucose was used for non-stimulatory conditions. Protocols that can be used for more specific discrimination of non-beta cells are described in the discussion section. (D) A typical trace of calcium dynamics of acinar cells stimulated by 25 nM acetylcholine. (E) A typical trace of calcium dynamics of ductal cells stimulated by 1 mM chenodeoxycholic acid. <a href="https://www.jove.com/files/ftp_upload/62293/62293fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After successful calcium imaging, the data are first exported and corrected for bleaching by a combination of an exponential and linear fit, as described in the protocol section. A time series before and after bleaching correction is presented in Figure 7A. Thereafter, several parameters in the activation and deactivation phase of the response as well as the plateau phase can be analyzed. A delay in the onset of [Ca2+]IC increase after stimulation can be measured as represented by delayA in Figure 7B and the heterogeneity in delays among individual cells (delayA1). The same parameters (delayD and delayD1)&#160;can be used to describe the deactivation phase. Following the initial transient [Ca2+]IC increase, the plateau phase in most pancreatic beta cells in an islet is characterized by relatively regular high frequency [Ca2+]IC oscillations. The plateau phase can be described by analyzing the classical functional parameters. The schematic presentation of [Ca2+]IC oscillations duration, frequency, and percentage of active time are presented in Figure 7C. In calcium imaging with acquisition rates higher than 10 Hz, calcium waves repeatedly spreading across the islet can also be recognized clearly (Figure 7C).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62293/62293fig07.jpg" /> 
Figure 7: Analysis of time-series data. (A) Correction of time-series data for photobleaching. (B) Analysis of delays to activation after stimulation and to deactivation after cessation of stimulation with 12 mM glucose. Duration of stimulation is denoted by the light gray, shaded bar in the image. (C) Analysis of several parameters of the plateau phase: I) Duration of the oscillation determined at half-height, II) frequency of oscillations determined by inter-oscillation intervals. III) active time as a product of frequency and duration of oscillations. I-IV) Delays between oscillations in any given wave of oscillations that spread across the islet of Langerhans determined by the delays (&#916;t) in time at which a single cell reaches half-height of the oscillation. <a href="https://www.jove.com/files/ftp_upload/62293/62293fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1. <a href="https://www.jove.com/files/ftp_upload/62293/Table 1 (3).xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices at JoVE.com

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

In combination with live cell calcium imaging, the acute pancreas tissue slice technique enables the study of intercellular waves and multicellular functional connectivity with a high resolution over long time periods. The main advantages of this technique are that it is fast, preserves tissue architecture, and reduces limited enzymatic and mechanical stress while maintaining a high yield of tissue. By detecting functional and morphological changes during disease progression, this technique helps our understanding of the pancreas related diseases, such as diabetes.This tissue slice technique can be applied to brain, pituitary, and adrenal tissues with an emphasis on the preservation of intercellular contact, paracrine interactions, and tissue architecture. To prepare for the injection of agarose into the mouse pancreas, fill a five milliliter syringe with 40 degrees Celsius liquid agarose and equip the syringe with a 30 gauge needle. After carefully covering the needle with a cap, place the syringe needle end down, with the entire volume of agarose below the water surface, in a 40 degrees Celsius water bath.Bubble a bottle of extracellular solution with carbogen, continuously on ice, at 1.5 milliliters per minute at barometric pressure and room temperature to ensure oxygenation and a pH of 7.4. To perform the agarose injection place a euthanized adult mouse under a stereo microscope and access the mouse abdomen via laparotomy. Gently move the gut to the left side to expose the common bile duct and use forceps to slightly lift the duodenal end of the duct to locate the papilla of Vater.Use a hemostat to clamp the bile duct at the duodenal papilla to prevent leakage of the agarose from the duct into the duodenum and use a small sharp forceps to reach under the common bile duct to break the membrane that attaches the duct to the pancreatic tissue. After clearing as much fat and connective tissue from the duct as possible, place the duct perpendicularly onto the tips of a pair of larger forceps, and pressing firmly on the syringe plunger, inject the viscous liquid agarose into the proximal end of the common bile duct for 20 to 30 seconds. When the tissue becomes whitish and slightly distended remove the syringe and slowly pour 20 milliliters of the bubbled ice-cold extracellular solution onto the pancreas to cool the tissue and to harden the agarose.To acquire pancreatic tissue slices use forceps and fine tough cut scissors to gently transfer the agarose injected pancreas into a 100 millimeter Petri dish containing 40 milliliters of ice-cold extracellular solution. Swirl the dish to rinse the tissue and transfer the pancreas into a second dish of ice-cold extracellular solution. Use the forceps and the scissors to cut the whitish, well-injected pancreas section into up to six 0.1 to 0.2 cubic centimeter pieces and remove any remaining connective and fatty tissue from the tissue pieces.Place the cleaned blocks into a 35 millimeter non-sticky bottom Petri dish containing approximately five milliliters of 40 degrees Celsius liquid agarose and immediately place the Petri dish on ice. When the agarose has hardened, hold the Petri dish upside down over the lid of a 100 millimeter Petri dish and use one half of a razor blade to carefully cut into the margin between the lateral wall of the Petri dish and the agarose to remove the agarose from the dish. Use the razor blade to cut individual cubes, each containing one tissue block of agarose, from the freed agarose and use cyanoacrylate glue to attach the blocks to the sample plate of a vibratome.Fill the cutting chamber of the vibratome with 150 milliliters of ice-cold extracellular solution continuously bubbled with carbogen. Screw fix the sample plate onto the vibratome and mount a new razor blade. Surround the chamber with ice and add two 10 milliliter volume ice cubes made with extracellular solution supplemented with six millimolar glucose.Set the slicer to 0.05 to 1 millimeter per second and 70 hertz, then begin slicing to acquire 140 micron thick agarose slices with a 20 to 100 square millimeter surface area. Use a fine paint brush to carefully transfer each slice into a 100 millimeter Petri dish filled with 40 millimeters of HEPES buffer supplemented with six millimolar glucose. For calcium dye preparation, dissolve 50 micrograms of cell permeable calcium indicator dye, 7.5 microliters of DMSO, and 2.5 microliters of the poloxamer, and 6.667 milliliters of HBS in a 15 milliliter screw cap tube.Use a pipette to thoroughly mix the solution for 20 seconds before submerging the tube in an ultrasonic bath chamber and vortexing, each for 30 seconds. Then aliquot 3.333 milliliters of the resulting calcium indicator dye solution into one 5 milliliter Petri dish per 10 slices of tissue to be labeled. For dye loading, use a thin soft paintbrush to transfer up to 10 tissue slices to each dish of dye solution and place the dishes onto an orbital shaker for 50 minutes at room temperature and 40 revolutions per minute protected from light.At the end of the incubation, transfer up to 20 stained slices into individual 60 milliliter Petri dishes filled with dye-free HBS. For calcium imaging by confocal microscopy select a 20 or 25 times magnification and set the acquisition mode to time-lapse imaging, the pinhole to 100 to 200 microns, and the excitation and emission for the fluorophore used in the experiment. Mount the recording chamber and perfusion system onto the temperature controlled stage of the microscope and position the inlet and outlet on the far edges of the recording chamber.Set the inflow and outflow rates to one to two milliliters per minute. Set the temperature of the profusion system to 37 degrees Celsius and initiate the profusion with the non-stimulatory solution. To record the calcium dynamics of the tissue place a single tissue slice into the recording chamber and immobilize the tissue with a U-shaped platinum weight with a taut nylon mesh.Use the bright-field option to locate the structure of interest and use live imaging to position the studied structures into the field of view. To optimize the signal-to-noise ratio, adjust the laser power, detector amplification, and line averaging binning while keeping the laser power minimal. Adjust the focal plane to 15 microns below the cut surface to avoid recording from potentially damaged cells and acquire the images.Setting the sampling frequency to one to two hertz to allow the initial detection of individual oscillations and recording a high resolution image. If available, use an online chart to obtain instant feedback on the preparation response, over illumination, photobleaching, and mechanical drift. When all of the images have been acquired, save the data for later analysis.Here an example of an optimal tissue slice can be observed in which the cell permeable calcium indicator dye was successfully loaded into the pancreatic tissue slice. In contrast, these tissue slices are not optimal for imaging due to either an unsuccessful dye penetration, a lack of islet cells, or an abundance of necrotic tissue on the samples surface. High resolution images of a pancreatic tissue slice can be used to delineate distinct regions of the pancreatic tissue, such as the islets of Langerhans, acinar tissue, or the pancreatic duct.Stimuli can be used to functionally discriminate between different islet cells or between islet and non-islet cells. For example, beta cells typically respond to a square pulse glucose stimulation by exhibiting a transient increase in intracellular calcium followed by a fast calcium oscillations on a sustained plateau. In contrast, non-beta cells respond with faster and more irregular oscillations.A delay in the onset of intracellular calcium increase after stimulation, as well as the heterogeneity and delays among individual cells can also be measured. The same parameters can be used to describe the deactivation phase. Here a schematic presentation of the intracellular calcium oscillation duration, frequency, and percentage of active time can be observed.When imaging at acquisition rates greater than 10 hertz, calcium waves that repeatedly spread across the islet can be clearly recognized. Clamp the common bile duct as closely to the duodenum as possible to prevent accidentally occluding the branch which is entering the pancreas. Also, do not stop depressing the plunger during the injection because the agarose will immediately harden in the needle.The acute mouse pancreatic tissue slice technique may also be used with the patch clamp method to study ion channel currents and access cytosis as well as immunohistochemistry studies and secretary studies.

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

Abstract