Axiocam hr rev3

Glass slides were prepared by placing four drops of petroleum jelly in a square arrangement and 50 μl of protoplasts at 1 M cells per ml in protoplast buffer within that square. A coverslip was then gently pressed down on the solution and jelly drops. Protoplasts were captured (Figure 3A) using a 40× objective on a Zeiss Axio Imager M1 fluorescence compound microscope fitted with an AxioCam HR Rev3 camera (Carl Zeiss Microscopy GmbH, Germany), with bright field and no filter or with UV light and a Filter Set 38 HE (Excitation filter BP 450–490 nm, Beam splitter FT 495 HE, emission BP 500–550 nm) to visualize lipid bodies stained with BODIPY^TM 493/503. Images were acquired using the Zeiss Zen imaging software (blue edition, Carl Zeiss Microscopy GmbH, Germany).

Body weight, body length, liver, inguinal, interscapular, and visceral epididymal fat pad weight were determined at 9 weeks of age. WAT and pancreas were fixed in 10% neutral buffered formalin, followed by staining of paraffin-embedded tissue sections with Heamatoxylin and Eosin (H&E) following standard procedures by the Medical Education and Laboratory Support Services (Memorial University). Images were acquired using an Axio Imager.A1 microscope with an AxioCam HR Rev3 using AxioVision software v 4.8.2.0 (Carl Zeiss Microscopy). WAT cell area was calculated using CellProfiler image analysis software [26 (link)]. Cell number was estimated from the mass of the WAT depots following the method of Jo et al. [27 (link)] except that volume was calculated from the measured area as described previously [28 (link)]. Pancreatic islet size was determined using ImageJ software [29 (link)].