Immunofluorescence Staining of MDSC Markers

TMAs were stained for IL-6, GM-CSF, and CD33⁺S100a9⁺ cells as a tissue-based surrogate of MDSC as published by Ortiz et al., with modifications.^{43 (link)} After rehydration and antigen recall in citrate buffer (Sigma Aldrich, St. Louis, MO, USA), slides were blocked with 5% BSA in PBS and incubated overnight at 4 °C with Abs to IL-6 (mouse 1:200, SantaCruz, Dallas, TX, USA), GM-CSF (1:100, Abcam), or S100a9 (rabbit 1:200, Abcam) and CD33 (mouse 1:100, SantaCruz) diluted in blocking buffer. Control slides were used to ensure that no background from primary or secondary Abs was present. After overnight primary incubation, slides were washed and incubated with secondary Abs to mouse (donkey anti-mouse 1:1000 Alexa 488, Abcam, Cambridge, UK) or rabbit (donkey anti-rabbit 1:1000 Alexa 568, Abcam) for 1 h at room temperature. Slides were stained with 4,6-diamidino-2-phenylindole (DAPI) at 1:5000 and washed, followed by 30-min incubation in 0.1% Sudan Black B in 70% ethanol to quench background fluorescence. Slides were washed with 0.02% Tween-20 in PBS, and coverslips were mounted using Vectashield Hardset (Burlingame, CA, USA). Images were taken using a Leica SP8 confocal microscope at ×20 (IL-6 and GM-CSF) or ×40 (CD33⁺S100a9⁺). Z-stacks were collected of CD33⁺S100a9⁺ cells, with 4 images collected over 3 µm.