Axio observer z1 fluorescent microscope

Paraffin-embedded pancreatic sections were incubated with primary antibodies followed by visualization using fluorescent secondary antibodies as previously published [16 (link)]. Primary antibodies: rabbit anti-insulin at 1:500 (Abcam, Cambridge, MA) and rat anti-bromodeoxyuridine (BrdU) at 1:1000 (Abcam). Secondary Abs: Cy-2 coupled donkey anti-rat IgG antibody at 1:200 (Jackson Immuno Research, West Grove, PA); Cy-3 coupled donkey anti-rabbit IgG antibody at 1:200 (Jackson Immuno Research). Nuclei were visualized with 1 μg/ml 4’,6-diamidino-2-phenylindole (DAPI, Life Technologies, Carlsbad, CA). Images of pancreatic sections were recorded with Axio Observer Z.1 fluorescent microscope with Axio Vision Imaging Software v 4.7.1.0 (Carl Zeiss, Oberkochen, Germany). Total beta cell area was measured via summation of insulin positive area in a pancreatic section with maximum footprint for each mouse using Mosai X (scan mode: meander, autofocus per tile) in Axio Vision. BrdU positive nuclei were manually selected if 1) it was surrounded by insulin and 2) superimposed with DAPI to pick up a proliferating beta-cell. The number of BrdU positive nuclei was corrected for total beta cell area of each mouse.

Gels were fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned. Sections were prepared for staining by deparaffinizing in a xylene substitute, rehydration, and heat-mediated antigen retrieval using pH 9 tris-EDTA with 0.05% tween 20. Sections were blocked in 10% goat serum and incubated with primary antibodies in a humidified chamber at 4 °C overnight. Secondary antibodies were added for 1 h at room temperature. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Antibodies were used at the following concentrations: anti-green fluorescent protein (anti-GFP) rabbit IgG Alexa Fluor 488 conjugated (1:75; Invitrogen A21311, Invitrogen, Carlsbad, CA, USA), rabbit polyclonal antibody to GJB1 [Cx32] (1:25; HPA010663, Sigma-Aldrich, St. Louis, MO, USA), biotinylated rabbit polyclonal antibody to red fluorescent protein (RFP) (1:500; Abcam, Cambridge, UK, ab34771), rabbit anti-laminin 1 + 2 (1:100; Abcam, ab7463), and mouse anti-laminin 5 antibody (1:100; Abcam, ab78286), Alexa Fluor 488 and 568 conjugated goat secondary antibodies (1:1000; Thermo Fisher Scientific) or avidin Alexa Fluor 488 (Thermo Fisher Scientific A21370). All sections were counterstained with DAPI and imaged by using a Zeiss axio-observer Z1 fluorescent microscope.

Immediately after removal from the needles, muscles were embedded in O.C.T. compound (Tissue Tek), frozen in liquid nitrogen-cooled isopentane and stored at −80°C, until analysis. 10 μm thick cryosections were cut with a cryostat (Thermo Electronic), maintained at −20°C and transferred onto static-free microscope slides. Immunofluorescent detection of myosin heavy chain isoforms was performed as previously described (Bloemberg and Quadrilatero, 2012 (link)). In addition, fiber membranes were visualized by staining for dystrophin (DSHB, University of Iowa, Iowa City, USA). Fiber type composition analysis was performed on image composites by counting all fibers across the entire cross section. Slides were visualized with an Axio Observer Z1 fluorescent microscope equipped with an AxioCam HRm camera and associated AxioVision software (Carl Zeiss, Germany).

Immediately after printing, the initial quantity of printed cells was verified by using manual counting and image analysis using ImageJ and Matlab. After printing, cells were monitored up to 21 days by using a Zeiss axio-observer Z1 fluorescent microscope. The size of organoids was determined by analyzing bright-field images taken daily for each experimental condition using ImageJ. Within this investigation, organoids were operationally defined as a cluster of cells with no clear cell–cell boundaries or the inability to discern individual cells from neighboring cells. All experiments were performed a minimum of three separate times. All quantitations presented in the results represent total observations across at least three independent experiments.