Image pro

Paraffin sections (4-µm thickness) were stained with hematoxylin and eosin (HE) for histological examination. For the immunohistochemical detection of insulin, paraffin sections were incubated with the rabbit anti-rat insulin antibody (cat#4590, Cell Signaling Technology, USA) at 1:100 dilution, followed by incubation with the HRP-conjugated anti-rabbit secondary antibody (cat#PV-9000, ZSGB-BIO, China) and development with the HRP-AEC kit (GENMED Scientifics, USA). Sections were then counterstained with hematoxylin and examined under the light microscope (Leica DM2000, Germany). For measurement of the islet area, islets in HE sections were identified and the islet area was measured using Image-Pro software (Media Cybernetics, USA). The average islet area from each rat was determined from 4-5 sections spaced >200 μm apart. The cell number per islet area in the same section was counted using Image-Pro (Media Cybernetics, USA) and the average cell number per islet from each rat was determined in the same manner as that of the islet area.

Western blotting was performed as previous reported [36 (link), 37 (link)]. Briefly, Hep-2 cells were grown in the mid-log phase, washed with phosphate buffered saline and lysed for 30 min on ice. The lysis buffer with pH 6.8 contained 10% glycerol, 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 62.5 mmol/L Tris-HCl. The SDS–PAGE analysis was performed and membranes were incubated overnight at 4°C with antibodies directed against the indicated proteins. The antibodies were obtained from the following companies: anti-TrkB, anti-β-actin, anti-p-Akt, anti-cyclin D1, anti-p-s-Src and anti-c-Src were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-RUNX3 and anti-Keap1 were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-Twist-1 and anti-Twist-2 were purchased from BD Biosciences (San Jose, CA, USA). Membranes were washed, incubated with the appropriate secondary antibodies and exposed with the ECL chemiluminescent substrate kit (Pierce, Rockford, IL, USA). Images were analyzed with ImagePro (Media Cybernetics, Bethesda, MD, USA) and densitometry data were analyzed by using either conventional Student’s t-test. Results are reported as mean ± SD. A P-value <0.05 was considered significant and all were two-tailed.

BMDM were stimulated as described above, supernatants were collected, and proteins were precipitated by methanol-chloroform extraction. Cell pellets were suspended in lysis buffer. Immunoblot analysis was performed using following primary antibodies: anti-mouse IL-1β (AF-401-NA; R&D System, MN USA), rabbit polyclonal anti-Caspase-1 (Santa Cruz Biotechnology), anti-β-actin (C4; Santa Cruz Biotechnology). For ASC oligomerization assay in lysates, cells suspension was washed with PBS and incubated with 2 mM disuccinimidyl suberate (DSS, No-Weigh™ Format, Pierce Protein Biology) for 30 minutes in room temperature. After washing with ice cold PBS, precipitates were suspended in lysis buffer. Immunoblot analysis was performed using rabbit anti-mouse ASC (N15; Santa Cruz Biotechnology). The supernatants were also used for anti-mouse ASC immunoblot and anti-phosphotyrosine (4G10; EMD Millipore, MA USA) immunoblot analysis. For immunoprecipitation, anti-phosphotyrosine agarose beads (PY20; EMD Millipore) were added into the supernatants and incubated for overnight, washed with lysis buffer for 3 times, and used for immunoblot with HRP conjugated rabbit anti-mouse ASC (LifeSpan Biosciences, Inc., WA USA). Image densitometry was performed using Image-Pro (MEDIA Cybernetics, INC., MD USA).

The experimental nests (e.g. Fig 1) were filmed through 200 h of observation time from a distance of 1.5 m with a high-definition (HD) camera (Panasonic HVX 200) and in parallel, with an infrared (IR) camera (Flir A320; resolution: 640 x 560 pixel; 9 Hz). The color-coded (rainbow-900 palette) IR images were transferred from the IMG-format (providing adjustable temperature scales when operated by Flir-Researcher 2.9) into the fixed-scale BMP-format, which was further analysed by the Image-Pro software (MediaCybernetics 7.0). In total, > 30 000 frames were selected for the final evaluation representing 120 min recording time. Selected images from both HD and IR films were processed by the ThermaCAM Reporter 9.1 Professional (Flir) and Image-Pro and superposed by triangulation to the view of the IR camera. This allowed the identification of the temperature values of bee bodies and of the cavities between.
Data loggers (HOBO U12) were mounted one meter in front and behind the nest and registered ambient humidity, irradiation and ambient temperature (T_amb), which latter was also used to calibrate the IR camera.