Orca er digital camera

Paraffin sections for pancreatic head and tail were prepared as previously described [30 (link),31 (link)]. The entire pancreas was sectioned every 200 μm, generating 8-16 sections for both head and tail pancreas. Primary antisera included guinea pig anti-insulin (catalogue number A0564, Dako, Carpinteria, CA, USA) and mouse anti-human Ki67 (catalogue number 550609; BD Biosciences, San Jose, CA, USA), followed by secondary antisera conjugated to Cy3 or Cy5 (catalogue numbers 706-166-148 and 715-605-151; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and DAPI (Molecular Probes, Eugene, OR, USA). EdU was detected by Click-iT EDU Alexa Fluor 647 Imaging kit (Invitrogen, Carlsbad, CA, USA). Images were acquired using Zeiss AxioImager (Carl Zeiss MicroImaging, Thornwood, NY, USA) with Orca-ER digital camera (Hamamatsu, Middlesex, NJ, USA). Slides were imaged to quantify beta cell morphometry as previously described [30 (link)], using Volocity 6.1.1 software (PerkinElmer, Waltham, MA, USA).

For cresyl violet motoneuron counts, 5 to 6 serial 40 μm thick sections
from lumbar 4 (L4) spinal cord segments separated by 800μm were stained as
described above. Spinal cord sections of 4 or 5 animals per group were examined. Images of
each ventral horn region were captured with an Axioscop light microscope equipped with an
Olympus camera DP71 and motoneurons were manually counted by a blinded operator.
Motoneurons were identified based on their location in the ventral horn, morphology as
well as presence of Nissl-positive staining in the cytoplasm. For immunostained
motoneurons counting, 10 to 12 serial sections stained as described above were used and
captured with an Olympus BX61 fluorescence microscope equipped with a Hamamatsu ORCA-ER
digital camera. Images were processed with the free NIH imageJ software.
For axonal count from Toluidine stain section, the whole ventral root area was
captured with a Zeiss Axioscop light microscope equipped with an Olympus camera DP71 and
images analyzed with the ImageJ software. GFP-positive axon counts were performed on
double labeled ventral and dorsal roots sections as described above. Three representative
fields were captured at 20x with a Zeiss laser-scanning confocal microscope and the total
number of axons and GFP-positive axons analyzed with the ImageJ software.

We collected the culture progeny of MuSCs from aged Myf5^nLacZ/+/Luciferase double-transgenic mice^{23 (link),43 (link)} by incubation with 0.1% trypsin in PBS for 2 min at 37 °C and transplanted them into tibialis anterior muscles of hindlimb-irradiated NOD/SCID mice. One month after transplant, we injected notexin to damage recipient muscles and activate MuSCs in vivo. Four days later, we collected, fixed, and cryosectioned recipient muscles, as described above. We performed immunohistological analysis of transverse tissue sections to detect β-galactosidase⁺ cells (indicating a donor-derived cell expressing Myf5, a marker of MuSC activation) in the satellite cell position within the myofiber basal lamina, as defined by laminin staining. We stained sections with anti-Laminin (Millipore, clone A5, catalog # 05-206, 1:250) and anti-β-galactosidase (Invitrogen, catalog # A11132, 1:100) primary antibodies and then with appropriate secondary antibodies (Invitrogen). We counter-stained nuclei with Hoechst 33342 (Invitrogen). We acquired images with an AxioPlan2 epi-fluorescent microscope (Carl Zeiss) with Plan NeoFluar 10×/0.30NA or 20×/0.75NA objectives (Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu). We captured digital images in OpenLab software (Improvision) and assembled them using Photoshop software (Adobe) with consistent contrast adjustments across all images.

All cell imaging experiments were performed using a Nikon Eclipse 80i epifluorescence microscope (Melville, NY) with a 60× 1.40 oil objective, and a Hamamatsu ORCA ER digital camera (Houston, TX) was used to acquire images. The MDA-1986 cells were seeded onto poly-L-lysine precoated glass coverslips (BD, Franklin Lakes, NJ) in 12-well culture plates at a density of 50,000 cells per well and allowed to grow overnight. Subsequently, cells were treated with PPa (6.48 μg/ml) or HA-ADH-PPa (6.48 ug/ml of PPa) in 1:5:94 (v/v/v) TWEEN® 80/DMSO/media followed by incubation at 37 °C for 5 h. The cell nuclei and lysosomes were stained with DAPI (10 μg/ml) for 5 min and LysoTracker® Blue DND-22 (4 μM) for 30 min, respectively. After three washes with 3 ml of PBS, the coverslips were placed on the slide glasses for imaging. The live cells were immediately imaged using a yellow fluorescent protein (YFP, ex/em, 500/535 nm) filter set (Nikon, NY) for imaging PPa and Ultraviolet excitation (UV-2E/C, ex/em, 360/400 nm) filter set (Nikon, NY) for imaging DAPI and LysoTracker® Blue DND-22 separately.