Ept through a mouthpiece during the process. Each iron dose was injected in 5 rats except20002000 which was injected in two groups of five rats by two researchers (M. Peek for for , g, which was injected in two groups of five rats by two researchers (M. PeekK. Saeki). This was completed to confirm reproducibility with the results betweenbetween reand and K. Saeki). This was completed to confirm reproducibility from the outcomes researchers, searchers, generating the total variety of for this experiment 55. Right after injection, injection, the creating the total number of rats employed rats utilised for this experiment 55. Immediately after the rats had been rats were placed back in their recovery. recovery. placed back in their cages for cages for Following 24 h, the rats were anesthetized and euthanized by cervical dislocation. In rats, Following 24 h, the rats had been anesthetized and euthanized by cervical dislocation. Inside the popliteal lymph nodes nodes will be the primary and Trometamol medchemexpress dominant SLNs draining the distal rats, the popliteal lymph would be the key and dominant SLNs draining the distal hindleg like the injection injection website. Following Aloisine A In Vivo euthanasia, SLND from the popliteal nodes hindleg such as the site. Following euthanasia, SLND on the popliteal nodes was performed bilaterally within a proneaposition. The caudalcaudal skin with the joint was incised, and was performed bilaterally in prone position. The skin in the stifle stifle joint was incised, the popliteal lymph nodes werewere anatomically located withinthigh muscles and disand the popliteal lymph nodes anatomically situated within the the thigh muscles and sected (Figure 1a).1a). Collected lymph nodes had been placedformalin. dissected (Figure Collected lymph nodes had been placed in in formalin. In selected animals from the 1000 (n = two), 2000 (n = four) and 4000 (n = 5) g iron injection In chosen animals = four) and 4000 (n = 5) iron injection groups, abdominal lymph nodes have been also harvested as secondary lymph nodes: Briefly, groups, abdominal lymph nodes were also harvested as secondary lymph nodes: Briefly, the animals have been placed inin dorsal recumbency, and celiotomy was performed.the cauthe animals were placed dorsal recumbency, and celiotomy was performed. At In the dal furcation of the descending aorta, iliaciliac lymph nodes were identified and resected. caudal furcation from the descending aorta, lymph nodes had been identified and resected. For all animals, the hindlegs were amputated bilaterally in the tarsal joints, weighed, For all animals, the hindlegs had been amputated bilaterally in the tarsal joints, weighed, and placed within a drying oven for 48 h h at 80 degrees Celsius. The dry weightthe distal legs a drying oven for 48 at 80 degrees Celsius. The dry weight of on the distal and was determined before powdering them using TissueLyser (20 Hz, 2 Hz, two min; Hilden, legs was determined before powdering them working with TissueLyser (20min; Qiagen,Qiagen, Germany), for further further The excised sentinel sentinel lymph nodes the powdered Hilden, Germany), foranalysis. evaluation. The excisedlymph nodes (SLN) and(SLN) and theCancers 2021, 13,four ofrat legs had been analyzed by SQUID to decide the volume of iron within the samples. The SLN uptake price was subsequently calculated as (Iron accumulation in SLN)/(Injected iron dose) 100. 2.2. Dilution and Time-Course Experiment Resovist equivalent to 200 iron (7.17 ) was diluted with saline two-, five-, and ten-fold, which resulted in final volumes of 14.34 , 35.84 , and 71.68 , respectively. All sa.
