Anti cd45 30 f11

Frozen skin tissue sections were incubated with different primary antibodies at 4 °C overnight; antibodies included anti-CD49f-biotin (GoH3, 1:150, BioLegend), anti-Ki67 (20Raj1, 1:100, eBioscience), anti-Akt (GTX28932, phospho Ser473, 1:150, GeneTex), anti-Akt (Akt 1+2+3) (GTX121937, 1:150, GeneTex), anti-CD34-biotin (RAM34, 1:150, eBioscience), anti-Lgr-5 (ab137484, 1:200, Abcam), anti-CD45 (30-F11, 1:150, BioLegend), anti-CD45-Biotin (30-F11, 1:150, BioLegend), anti-F4/80-Biotin (BM8, 1:150, BioLegend), anti-F4/80 (BM8, 1:150, BioLegend), anti-Ly6C-Biotin (RB6-8C5, 1:200, BioLegend), anti-Ly6C (HK1.4, 1:200, BioLegend), anti-MHC II (14-4-4S, 1:200, eBioscience), anti-TNF-α (1F3F3D4, 1:100, eBioscience), anti-TNFR1-Biotin (55R-170, 1:100, BioLegend), anti-β-catenin (C2206, 1:150, Sigma) and anti-Phospho-β-Catenin (Ser552) (5651S, 1:100, CST Signaling). Secondary antibodies with different fluorescence conjugates (FITC, Cy3/TRITC and Alex Fluor 647) were used for detection, and cells were visualized with an Olympus FV1000 confocal microscope.

To isolate intestinal epithelial cells, colonic tissues were incubated with 1 mM EDTA in PBS at 37°C for 20 min, and the single-cell suspensions were prepared by mechanical dissociation with vigorous shaking in PBS. The suspensions of epithelial cells were stained with anti-CD45 (30-F11) and anti-EpCAM (G8.8; both from BioLegend), followed by propidium iodide (PI). PI⁻CD45⁻EpCAM⁺ fraction was sorted to 90–95% purity with BD Aria III by using a 130-μm nozzle, and RNA was isolated using the RNeasy kit (QIAGEN). cDNA was amplified using a NuGen WT Ovation Pico kit and hybridized to an Affymetrix GeneChip (Mouse Genome 430 2.0 Array). The data were analyzed with GeneSpring12.5 (Agilent) and DAVID (Database for Annotation, Visualization, and Integrated Discovery; Huang et al., 2009 (link)).

Colon cells were stained with Fc Block (Clone 2.4G2) followed by staining with fluorescently labelled antibodies in IEC buffer (PBS with 5% FBS and 2 mM EDTA to reduce cell clumping) for IECs or 2% FBS in PBS for T cells on ice in 1.5 ml microcentrifuge tubes. For intracellular staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm kit (BD Bioscience). Samples were acquired on an Attune NxT flow cytometer (Life Technologies) and analysed with FlowJo software. Cells were sorted on either a BD ARIA II or S6 Enceladus (BD Biosciences). The following antibodies/reagents were used: anti-CD45 (30-F11; Biolegend), anti-EPCAM1 (G8.8; ThermoFisher), anti-FABP2 (polyclonal; R&D/Fisher), anti-goat IgG (ThermoFisher), anti-LY6G (1A8; BioLegend), anti-MHCII (M5/114.15.2; Fisher), Live/Dead Fixable Near-IR dead cell dye (ThermoFisher), anti-CD4 (RM4-5; BioLegend), anti-TCRβ (Η57−597; ThermoFisher), anti-CD44 (IM7; BioLegend), anti-CD45.1 (A20; ThermoFisher), anti-CD45.2 (104; BD Biosciences), anti-IL-17A (TC11-18H10; BD Biosciences), anti-IFNγ (XMG1.2; ThermoFisher), and anti-IL-22 (1H8PWSR; ThermoFisher).

1 × 10⁶ B16F10 melanoma cells were injected into the flank of C57BL/6 RAG2^−/− mice (8–12 weeks old). Twelve days later the growing tumors were collected and processed for transplantation into the flank of WT C57BL/6 mice (8–12 weeks old; Harlan). Vaccination was carried 5 days later as described in Table 1.
Tumors were collected 14 days later as the control groups reached the humane end-point of 1,000 mm³. Tumor volume was measured using a caliper according to the formula V = (W × W × L)/2, where V is tumor volume, L is tumor length and W is tumor width. TILs were isolated as described earlier. TILs were stained with anti-CD8α (53–6.7, BD Biosciences), anti-IFN-γ (XMG1.2, BD Biosciences), anti-CD45 (30-F11, Biolegend), anti-Ly6G (1A8, Biolegend), and anti-Ly6C (HK1.4, Biolegend) for flow cytometric analysis by FACSCanto and FlowJo Software. Survival was evaluated using the same designated vaccines and the transplanted WT C57BL/6 mice (8–12 weeks old; Harlan) were vaccinated twice a week and tumor size was monitored daily and measured using a caliper.

Cells were stained with fluorochrome-conjugated mAbs for 30 min on ice in the presence of Fc block (anti-CD16/CD32; clone 2.4G2, BioXcell) and normal rat serum (Jackson). Staining and washing was performed in FACS buffer containing PBS, 0.1% BSA, and 1 mM EDTA. The following antibodies were used: anti-CD11b (clone M1/70; BioLegend), anti-P2X7 (clone RH23A44, UKE), anti-CD45 (30-F11, Biolegend), anti-CD4 (clone RM4-5; BioLegend), anti-CD8a (clone 53–6.7, Biolegend), anti-CD25 (clone PC61, Biolegend) and anti-FcεR1α (clone MAR1, Biolegend). Cells were analysed using a BD Celesta flow cytometer and data were analysed with FlowJo software (Treestar).

Brains were isolated from mice perfused transcardially with PBS and 2% PFA. Brains were cut sagittally and placed in 2% PFA overnight, and subsequently placed sequentially in a series of sucrose solutions of increasing concentration (10%, 20%, and 30%). Murine brains were frozen in optimal cutting temperature (O.C.T.) compound (Tissue-Tek, Tokyo, Japan) in hexane pre-chilled in liquid nitrogen. Frozen brain sections (8-9 μm) were fixed in methanol, rinsed in tris-buffered saline with 0.05% Tween 20 (TBST) and blocked with 10% FCS. Excess solution was shaken off before primary fluorophore-conjugated antibodies (anti-CD11b: M1/70, Biolegend and anti-NS1: 4G4, Roy Hall, UQ) were applied. Regions of interest were selected based on immunofluorescent staining visualised on the Olympus BX-51 microscope using a DP-70 camera and Cell Sensor software. Slides were then incubated with metal-conjugated antibodies overnight at 4 °C (anti-Ly6C (HK1.4, Biolegend), anti-CD86 (GL1, Becton Dickinson), anti-Iba1 (EPR165, Abcam), anti-CD45, (30-F11, Biolegend), anti-FITC (FIT-22, DVS) and anti-NeuN (Fox3, Biolegend)). Iridium DNA intercalator (Fluidigm, 1:2000 in TBST) was applied to brain sections, prior to sections being rinsed in UltraPure water and left to air dry. If slides were not immediately ablated on the Hyperion, they were stored at 4 °C until use.