Digital eclipse c1 microscope

HeLa cells grown on coverslips were co-transfected with mCherry-P58^IPK along with either Myc-NP or Myc-NP mutants for thirty-six hours using TurboFect Transfection Reagent. Cells were washed once with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The excess paraformaldehyde was neutralized with 0.1 M glycine in PBS, followed by extensive washing with PBS. The fixed cells were permeabilized with 0.5% TritonX-100 in PBS for 5 min and blocked with 3% BSA for 2 hours at room temperature. The cells were sequentially incubated with anti-Myc and Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) antibody (life technologies). The coverslips were mounted in mounting medium containing DAPI (vector labs). The confocal images were recorded with Nikon Digital Eclipse C1 Microscope.

Tumor xenografts were fixed in 4% neutral formalin at room temperature for 24 h, embedded in paraffin and cut into 4 μm sections. The sections were then deparaffinized in xylene, rehydrated in a graded alcohol series and treated with boiling 0.01 mol/l citrate buffer for 15 min for antigen retrieval. Endogenous peroxidase activity was blocked with hydrogen peroxide (0.3%) at room temperature for 15 min, and the sections were incubated with 5% bovine serum albumin (R&D Systems, Minneapolis, MN, USA) at 37 °C for 45 min to reduce non-specific binding. Immunostaining with MET antibody (#8198; Cell Signaling Technology) or Ki-67 (ab15580; Abcam, Cambridge, UK) were carried out at 4 °C for 16 h, followed by incubation with a horseradish peroxidase (HRP) -conjugated secondary antibody from the Envision kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 45 min at room temperature. Antibody binding was detected by DAB (Dako; Agilent Technologies, Inc.), according to manufacturer's protocol, at room temperature for 1 min and the reaction was terminated by immersion of tissue sections in distilled water once brown staining appeared. Tissue sections were counterstained with 1% hematoxylin at room temperature for 3 min and dehydrated in a graded series of ethanol. Images of representative fields were obtained from Nikon Digital ECLIPSE C1 microscope (Nikon Corporation).

D. melanogaster wings were visualized using a Nikon Digital Eclipse C1 microscope (Nikon; Tokyo, Japan). Every 10 days, for a period of 50 days, flies were anaesthetized with CO₂, and their wings were carefully dissected and mounted with DePeX (Serva Electrophoresis GmbH, Heidelberg, Germany) onto glass microscope slides. The morphology and structure of the wings were observed separately for each gender. Wing surface area was measured using ImageJ v1.54d software.