2.4.1 Immunocytochemistry
The immunostaining of insulin- and glucagon-containing islet cells was performed using the following standard protocol: First, fresh slices were permeabilized and fixed by incubation in 4 % paraformaldehyde and 0.3 % Triton X-100 in phosphate buffered saline (PBS) for 30 min at room temperature. After repeated washing steps (3×5 min in PBS) the primary anti-insulin (mouse, monoclonal) and anti-glucagon (rabbit, polyclonal; Dako, USA) antibodies were added at dilutions of 1:1000 and 1:500 in PBS and 3% bovine serum albumin (BSA). After 2 h of incubation at 37°C(or alternatively overnight at 4°C) and 5 min of washing in PBS the secondary antibodies Alexa 488 (goat anti-mouse, Molecular Probes, USA) and Alexa 647 (goat anti-rabbit, Molecular Probes, USA) at a dilution of 1:500 in PBS were added for 45 min at 37°C. After additional washing in PBS the slices were mounted on microscope slides with the SlowFade Light Antifade Kit (Molecular Probes, USA) to prevent bleaching of the specimen.
Images of the stained islet cells were obtained using a confocal microscope (TCS SP2, Leica, Germany). The fluorophores were excited using laser lines of 488 nm (Ar) and 633 nm (He-Ne). Emission was detected at 505–530 nm (green channel), and 656 nm (red channel).
2.4.2 Ca2+-imaging
Single-cell Ca2+–imaging
The water soluble K+-salt of Fura-PE3 (TEF Labs, USA, 50µM added to the pipette solution) was used to monitor intracellular Ca2+ concentration changes (∆[Ca2+]i) simul-taneously with the patch-clamp recordings. Fura-PE3 was used because it is less prone to compartmentalization and leakage from cells than the commonly used Fura-2 while still showing identical spectral characteristics (Quigley et al.,1995). Monochromatic light (Polychrome IV; TILL Photonics, Germany) alternating between 340 and 380 nm (50 Hz) was short-pass filtered (at 410 nm), reflected by a dichroic mirror (centered at 400 nm) and directed through a 60×water immersion objective (CFI Fluor, N.A. = 1) focussed on the vertical midline of the patched and loaded cell. The emitted fluorescence was transmitted by the dichroic mirror and further filtered through a 470 nm barrier filter. Contrast enhancing elements like the DIC prism were removed from the light path to obtain optimal transmission conditions. Images were obtained using a cooled front-illuminated frame-transfer emCCD camera (Ixon, Andor Technology, Japan) and native Andor software. A subarea of 256×128 pixels of the CCD chip was illuminated for 6 ms per wavelength and 2×2 on-chip binning was performed to reduce image noise and achieve sufficient acquisition speed.
[Ca2+]iwas calculated from the background subtracted intensity ratios of the images obtained with 340 and 380 nm excitation using the equation derived byGrynkiewicz et al.(1985):
[Ca]free=K∗d× R−Rmin
Rmax−R (2.1)
A 3-point in situcalibration was performed by dialyzing β-cells with IS-2 of low (∼0µM), medium (0.367µM) and high (∼36µM) free Ca2+ concentrations to obtain the fluorescence ratios Rmin, Rdef and Rmax, respectively. For low Ca2+ 10 mM of K-EGTA was added and high Ca2+ was obtained using 10 mM of Ca-EGTA. To achieve a sufficiently high Ca2+ concentration, all calibration solutions contained 1 mM ATP instead of 4 mM to reduce the buffering effect of ATP. The defined Ca2+ concentration was achieved by mixing Ca-EGTA and K-EGTA at a ratio of 2.¯3 :1. Free Ca2+ was
2 Material
calculated using the MaxChelator software (Patton et al.,2004). The apparentKdfor the experimental conditions used in this study was calculated according to
K∗d =[Ca]def× Rmax−Rdef
Rdef−Rmax (2.2)
Thein situcalibration was performed using 2–3 cells for each calibration solution.
The resulting apparentKdwasK∗d =1.2 µM and Rminand Rmaxwere determined to be 0.14 and 0.98, respectively.
All necessary calculations were performed using a custom written Matlab (MathWorks Inc., USA) script and the image acquisition and hardware triggering parameters were calculated and controlled by a custom AndorBasic (Andor Technology, Japan) program.
In addition to monitoring [Ca2+]i, images of the dye loaded cells were used for the morphometric analysis of cell size to complement the resting membrane capacitance (Cm) measurements (see below, section2.5). The β-cell cross-sectional area (A) of a custom-shaped region of interest (ROI) enclosing the cell perimeter of the midline focus section was determined with the WiseVision software (Wise Technologies, Ljubljana, Slovenia). The total surface area (F) of the cell was approximated by assuming spherical cell shape according to
A = πr2 (2.3)
F = 4A (2.4)
The obtained images were size-calibrated using images of fluorescent microspheres (4 µm, Molecular Probes, USA) excited at 380 nm.
Islet Ca2+-imaging
To observe glucose-induced∆[Ca2+]i in a large number of islet cells, slices were bulk loaded with the acetoxymethyl (AM) ester of Fura-PE3 (Stock: 4 mM in DMSO with 5% pluronic acid F-127, Molecular Probes, USA). Fresh slices were loaded with Ca2+-indicator by incubation in standard extracellular solution containing a final con-centration of 6µM Fura-PE3-AM. After loading for 60 min on an orbital shaker, the slices were incubated for at least 15 min in indicator-free extracellular solution at 32°C
to achieve a sufficient degree of deesterification. The final concentrations of DMSO and pluronic acid in the loading solution were 0.15% and 0.007%.
A similar setup was used for islet imaging as for single cell Ca2+-imaging except that a 20×water immersion objective (CFI Fluor, N.A. = 0.5) was used and ratio image pairs were obtained at a frequency of 1 Hz (exposure time: 60 ms per image). Images were obtained from medium sized islets at a focal plane well below the first layer of cells close to the islet center. The excitation light intensity was reduced by two neutral density filters (ND8, ND4) to minimize phototoxic effects resulting from UV-exposure as much as possible. Furthermore, excitation light was constantly shut offexcept during periods of CCD exposure. Images were 512×512 pixels in size and 2×2 on chip binning was performed to reduce noise and file size. The images taken during 340 and 380 nm excitation were background corrected by subtracting the average pixel intensity of tissue free areas from the intensity values of all image pixels for each frame of the time-lapse recording separately. The F340/F380ratio images presented in this work have been subjected to a Gauss filter (mask size: 5×5 pixel,σ =2) and noise was further reduced by 4× temporal binning. Mean pixel values from ROIs of the F340/F380 images were obtained from the raw data before any image processing was performed. Image analysis and acquisition were done using the Andor software in connection with custom written AndorBasic scripts. Because actual [Ca2+]i levels were of no interest in this particular experiments, no attempt was made to calibrate the fluorescence intensity ratios.
All experiments were performed under constant perfusion of the recording chamber with carbogenated extracellular solution at 37°Cat a constant flow rate of∼1.5 ml min-1. The delay time for a solution change to reach the recording chamber ranged form 40–45 s. All data was adjusted accordingly.