Cd11b apc clone m1 70

Tumors were carefully excised with adequate margin from the mice on day 24 as illustrated in Figure 2A, embedded in Tissue-Tek OCT compound (Sakura, 4583), and promptly frozen on dry ice. The OCT-embedded tumors were then sectioned into 12μm slices using a cryostat. These sections were fixed with ice-cold pure acetone for 10 min and air-dried for an additional 15 min. To ensure optimal preparation, the sections were prewetted three times in PBS for 5 min each. Subsequently, slides were blocked with 1% BSA and 1% normal mouse serum in PBS for 1 h. Following the blocking step, the sections were directly stained with a CD11b antibody (CD11b, APC, Clone M1/70, BD Biosciences) diluted to a concentration of 1:500 in the blocking buffer. Staining was carried out for 1 h, followed by three washes with PBS for 5 min each. Finally, sections were mounted with a single drop of Fluoroshield with Dapi (Merck, F6057), and images were captured using a Zeiss Axio Imager.A2 microscope.

Cell suspensions were made and cells stained as described (25 (link), 37 (link)) using the following directly conjugated antibodies from BioLegend unless otherwise stated: CD11b APC (clone M1/70), Siglec-F PE (BD Bioscience, clone E50-2440), CD4 FITC, AF700 (clone GK1.5), ST2 APC (clone DIH9), CD117 APC (ACK2), FcεRI PerCP-eFluor 710 (eBioscience, clone MAR1), CD49b PE (clone DX5), IL4rα PE (clone I015F8), and CD25 (clone 3C7). A minimum of 1 × 10⁵ cells were acquired, and doublets were excluded by gating cells on FSC-H/FSCA, as illustrated in Supplementary Figure 1. For intracellular cytokine staining, mLN cells were stimulated with Cell Activation Cocktail (with Brefeldin A, BioLegend) at 1 × 10⁶ cells/ml, incubated in complete RPMI (10% FBS and 5% Pen/Strep) at 37°C for 6 h. Then, cells were fixed using Ic Fixation buffer (Invitrogen) and permeabilized using Permeabilization Buffer (Invitrogen) to carry out the intracellular staining for IL-4 PE (clone 11B11), IL-13 Pecy7 (Invitrogen, clone eBio13A), and IFN-γ FITC (clone XMG1.2). Cells (2 × 10⁶) were used for transcription factor staining. Staining against Foxp3 PE and Gata3 PE was performed using the Transcription Factor Staining Buffer Set according to the manufacturer’s instructions. Data were acquired on a C6 Accuri flow cytometer (BD Biosciences) or Cytoflex (Beckman) and analyzed using FlowJo v10.6.