Lmd6000 microscope

Eyes extracted from three rats in each age group were processed for embedding and sectioning using standard protocols, as described previously [19 (link),26 (link)]. One eye of each rat was used for morphological evaluation, while the fellow eye was used for immunohistochemistry. Eyeballs were fixed in 4% paraformaldehyde in phosphate-buffer (pH 7.2), followed by embedding and sectioning for paraffin or cryosections. While the sections stained with hematoxylin and eosin were analyzed by light microscopy for retinal morphology, the ultrastructure was analyzed by electron microscopy. Immunohistochemistry on retinas of 12-month-old rats was performed on paraffin sections. The transverse sections (4 μm) were mounted in silane coated slides. Deparaffinized sections were processed in 10 mM sodium citrate buffer (pH 6.0) and heated for antigen retrieval. After blocking, the respective primary antibodies were added to the sections in a volume of 100 μl and allowed to incubate overnight at 4 °C. After incubation, the sections were washed three times for 5 min in PBS, and 100 μl of respective Alexa Fluor-555 and Alexa Fluor-488 secondary antibodies solution was added. After washing, the slides were mounted in anti-fade reagent containing DAPI and observed under a Leica LMD 6000 microscope (Wetzlar, Germany). Images were captured using appropriate filters.

All equipment, including microtome, blades, water bath, brushes, and polyethylene naphthalate membrane (PEN-membrane) slides (Applied Biosystems, Foster City, Calif), was sprayed with RNase Away (ThermoFisher) before tissue sectioning. Tissue sections were sliced at a 12-μm thickness (separated by 36 μm), deparaffinized, rehydrated, and stained with hematoxylin and eosin. Slides were air dried, followed by brief incubation on a hot plate and immediately taken to the Leica LMD6000 microscope (Buffalo Grove, Ill) for islet isolation as previously described.^{37 (link)}

Paraffin-embedded islet grafts (n = 8) were consecutively sectioned, and every other section was stained for insulin (guinea pig polyclonal; dilution 1:400; Fitzgerald, Acton, MA), detected by MACH 3 Rabbit HRP-Polymer Detection (Biocare Medical, Concord, CA) and visualized by 3,3′-diaminobenzidine.^{36 (link)} The insulin area was determined manually using a Leica LMD6000-microscope (Leica Microsystems, Wetzlar, Germany). The total area of the islet graft was calculated as an area under the curve by plotting the known areas.
Sections were double-stained for insulin (Fitzgerald) and glucagon (mouse monoclonal, dilution 1:800; Abcam, Cambridge, UK) with Alexa Fluor 488 (goat anti-guinea pig) and Alexa Fluor 594 (donkey antimouse; dilution 1:250; Invitrogen, Carlsbad, CA) as secondary antibodies. Images were acquired with Zeiss LSM780 (Zeiss, Jena, Germany) confocal. The area of insulin and glucagon was determined with the image software Imaris (Bitplane AG, Zurich, Switzerland). The estimated total α cell area was calculated by multiplying the α cell percentage with the total insulin area for the respective islet graft.