Ax 51 epifluorescent microscope

The AQUA method of QIF has been described elsewhere [29] (link). This method allows exact and objective measurement of fluorescence intensity within a defined tumor area, as well as within subcellular compartments. Briefly, a series of high-resolution monochromatic images were captured using an Olympus AX-51 epifluorescent microscope based on a previously described algorithm for image collection [29] (link). According to this algorithm, images were obtained for each sample histospot and for each different fluorescence channel, DAPI (nuclei), Alexa 546 (cytokeratin), or Cy5 (target probe), respectively. A tumor mask was created by binarizing the cytokeratin signal to distinguish stromal from tumor area and target probe expression was quantified only in the tumor. AQUA scores were calculated for a given target within the “tumor mask” by dividing the signal intensity by the area of the “tumor mask” within each histospot. Histospots containing less than 5% tumor, as determined by the percentage of area which was positive for cytokeratin were excluded from the analysis.

Oxytocin-positive and vasopressin-positive cell bodies were counted using Olympus AX51 epifluorescent microscope with FITC (for retrograde label) and Texas Red filters (for oxytocin and vasopressin) with the experimenter blinded to the experimental groups. Retrograde tracer injection into the RVLM predominantly labels the ipsilateral side of the PVN^{16 (link),18 (link)} and so quantification was carried out only on the ipsilateral side of the pPVN to the retrograde tracer injection site. The number of cells with either retrograde-label, oxytocin-positive, or vasopressin-positive cells were counted, as well as the number of cells that co-expressed retrograde label and either oxytocin or vasopressin.