Dp manager software

Tissues were fixed in 4% paraformaldehyde overnight at 4 °C, embedded in paraffin, sectioned and stained with periodic acid-Schiff. Slides were evaluated using an Olympus BX51 microscope, and image acquisition was conducted with the Olympus DP70 camera and DP-Manager software. The percent area of goblet cells and colonic hyperplasia were calculated using a technique established previously51 (link).

A-673 Ewing’s sarcoma cells were treated by 20 μmol/L ZA [1-hydroxy-2-(1H-imidazole-1-yl) ethylidene-bisphosphonic acid supplied as the disodium salt by Novartis Pharma AG] during 24 h. Invasion of cultured cells was analyzed using Boyden’s chambers (8 μm pores, Becton Dickinson Labware) covered by polyethylene terephtalate membrane with Matrigel® coating (2 μg/100 μL/well in cold PBS) in 24-wells plate (Multiwell™ 24, FALCON®). At the end of the 24 h-period, viable cells were counted (by trypan blue exclusion) and the same number of ZA treated and non treated viable cells (4.10⁴) were seeded in the upper compartment of 500 μL cups in 1% FBS medium. The chamber was immerged in 700 μL of 10% FBS medium and left 48 h for incubation at 37°C in 5% CO₂ humidified atmosphere. Non invasive cells were removed and invading cells present on the inferior surface of the membrane were fixed by 3% PFA (ParaFormAldehyde) and stained by methylene blue. After drying, the invasive cells were counted with 10× microscope in 5 microscopic fields using DP controller, DP manager software (Olympus). All experiments were repeated 3 times in duplicates and invasion is expressed by mean number of cells / field.

Cells seeded on coverslips were fixed in 4% paraformaldehyde in DPBS at RT followed by permeabilization with 0.5% of Triton X-100 (Santa Cruz Biotechnology, Dallas, TX, USA) in DPBS 1x (PBTx) at RT for 5 min. Fixed cells were then blocked with 1% Bovine Serum Albumin (BSA) (Sigma-Aldrich) and 5% Normal Donkey Serum (NDS) (Sigma-Aldrich) in PBTx for 30 min at RT in darkness and incubated with primary antibodies diluted in blocking solution overnight at 4 °C in darkness. After primary antibody washing, secondary antibodies diluted in blocking solution were incubated for an hour at RT in darkness. Cells were stained and mounted on microscope slides using Fluoroshield with DAPI (Sigma-Aldrich). Immunostaining was performed using the following primary antibodies: rabbit anti-vimentin (1:500; Proteintech, Manchester, UK), mouse anti-alpha smooth muscle actin (α-SMA) (1:1000; Abcam) and mouse anti-myosin 4 (1:500; Invitrogen). Secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (ThermoFisher Scientific, Waltham, MA, USA). Immunofluorescence analysis was performed using Olympus BX51 Fluorescence Microscope (Olympus) and cell images were acquired by DPController and DPManager software (Olympus).

The analysis and photo documentation of slides was performed using bright-field light microscopy (Olympus BX51) by the program DP Manager Software (Olympus). In order to define the morphological pattern of each experimental group and guarantee a high-accuracy histopathological diagnosis, six slides each with 12 nonsequential histological sections at different depths were made for each individual (n = 5 per group), which resulted in 72 analyses per individual and 360 for each experimental group [25 (link)]. On average, 125 images/group were documented, and it was possible to observe the dorsal vessel, lumen, pericardial cells, trophocytes, oenocytes, and immune system cells.