Cat 15710

Skin was harvested for evaluation at two different timepoints. The first set of mice was sacrificed following completion of IR to assess the immediate effects of IR and prophylactic DFO on the skin. At this timepoint, because the IR only and DFO post-IR groups are equivalent (having received irradiation but no DFO treatment), half (4/8) of the mice were sacrificed from the IR only group. Half (4/8) of the mice in the DFO ppx group were also sacrificed at this time for early analysis. Tissues were harvested immediately after the conclusion of IR for ROS assays at the time where oxidative stress is highest during and within hours after radiation insult^{61 (link),62 (link)}. Scalp skin was either fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Cat#15710) at 4 °C for 18 h, snap frozen in OCT, or directly stored at − 80 °C for histological analysis, ROS detection, or protein quantification, respectively. The remaining mice across all four groups (n = 4/group) were sacrificed six weeks after the completion of IR to assess chronic effects of IR on the skin^{4 (link)}. Scalp skin was fixed in 4% PFA at 4 °C for 18 h and similarly processed for histological analysis.

Single-cell suspension from the BM of the experimental and control mice were prepared, followed by RBC lysis, and fixed with 2% PFA (Electron Microscopy Sciences Cat15710) at 37 °C for 10 min. Cells were washed twice with PBS and then slowly permeabilized with ice-cold 95% MeOH while vortexing on ice for 20 min. Cells were washed twice with PBS and incubated with Fc-block for 15 min on ice and then stained with PE/Cy7-conjugated anti-B220, PE-conjugated anti-CD43, AlexaFluor 647-conjugated anti-phospho-STAT5 (pY694, BD Biosciences), or mouse IgG₁,κ isotype control (BD Biosciences). PE/Cy7-conjugated anti-B220 and PE-conjugated anti-CD43 were used at dilution of 1:400. AlexaFluor 647-conjugated anti-phospho-STAT5 was used at dilution of 1:100.