Six tumor-bearing rats were assigned to be either sedentary or exercising, with three rats in each cohort. Individuals selected to be tumor-bearing subjects were administered flank injections with MTLn3 rat mammary adenocarcinoma cell line 4 weeks after placement in cages. After injection, tumor growth was monitored and the voluntary wheel-running of exercising rats was recorded using cycle counters. Twenty-one days post-injection, or when tumors grew to three centimeters, rats were euthanized using sodium pentobarbital.
Following euthanization, spleen and tumor tissue was excised from rats. Removed tissue samples were cut into small pieces, placed in cryomolds, coated in OCT compound, and frozen with isopentane and liquid nitrogen. Cryomolds were stored at -80°C. From storage, tissue samples were cryosectioned to 4 microns at 20°C. Cryosectioned tissue was placed onto 8 poly-D-lysine-coated slides. Each slide consisted of 2 tumor samples and 2 spleen samples. 1 tumor sample and 1 spleen sample on every slide was designated to be the negative control while the remaining 1 tumor sample and 1 spleen sample were designated to be stained. 2 slides were cut for each rat.
Tissue was fixed in acetone at -20°C in order to prevent the dissociation of the tissue. Tissue samples were then washed with TBS (0.05% Tween 20 in PBS) to help dissociate the cell membranes. Permeabilization of the membranes was accomplished utilizing a detergent comprised of Tween 20 and 0.1% Triton X-100. The blocking buffer, 1% NGS in PBS was applied to the tissue samples to increase antibody affinity. Positive control tissue samples were then stained with antibodies for CD25 and Foxp3 at a concentration of 1:200 for 1600 microliters. After being held at 4°C for 1 hour, the samples were washed in TBS. Hoechst dye was applied to all tissue samples for 10 minutes to stain the cell nuclei. Cover slips were fitted using 50% glycerol.
The tissue samples were visualized using confocal microscopy to distinguish CD25 and Foxp3 and therefore identify Tregs. For each sample, 5 fields of view were randomly selected and observed in 40x magnification. Images were translated into quantitative data by counting the number of Tregs present in each of the positive control tissue samples. T tests were run, comparing overall tumor and spleen samples, spleen samples between cohorts, tumor samples between cohorts, and overall tissue samples between cohorts, in order to analyze the data and determine the statistical significance of the findings.
Following euthanization, spleen and tumor tissue was excised from rats. Removed tissue samples were cut into small pieces, placed in cryomolds, coated in OCT compound, and frozen with isopentane and liquid nitrogen. Cryomolds were stored at -80°C. From storage, tissue samples were cryosectioned to 4 microns at 20°C. Cryosectioned tissue was placed onto 8 poly-D-lysine-coated slides. Each slide consisted of 2 tumor samples and 2 spleen samples. 1 tumor sample and 1 spleen sample on every slide was designated to be the negative control while the remaining 1 tumor sample and 1 spleen sample were designated to be stained. 2 slides were cut for each rat.
Tissue was fixed in acetone at -20°C in order to prevent the dissociation of the tissue. Tissue samples were then washed with TBS (0.05% Tween 20 in PBS) to help dissociate the cell membranes. Permeabilization of the membranes was accomplished utilizing a detergent comprised of Tween 20 and 0.1% Triton X-100. The blocking buffer, 1% NGS in PBS was applied to the tissue samples to increase antibody affinity. Positive control tissue samples were then stained with antibodies for CD25 and Foxp3 at a concentration of 1:200 for 1600 microliters. After being held at 4°C for 1 hour, the samples were washed in TBS. Hoechst dye was applied to all tissue samples for 10 minutes to stain the cell nuclei. Cover slips were fitted using 50% glycerol.
The tissue samples were visualized using confocal microscopy to distinguish CD25 and Foxp3 and therefore identify Tregs. For each sample, 5 fields of view were randomly selected and observed in 40x magnification. Images were translated into quantitative data by counting the number of Tregs present in each of the positive control tissue samples. T tests were run, comparing overall tumor and spleen samples, spleen samples between cohorts, tumor samples between cohorts, and overall tissue samples between cohorts, in order to analyze the data and determine the statistical significance of the findings.