Brilliant violet 605 anti mouse cd69

Lymphocytes from blood, spleen, and bone marrow of immunized mice were harvested on day 7 after the final immunization and analyzed by flow cytometry. Dead cells were excluded by viability dye staining, and adherent cells were excluded by SSC/A and SSC/H gating analysis. Cells were analyzed by a BD LSRFortessa™ Flow Cytometry (BD Biosciences, USA). Data were acquired and analyzed by FlowJo version 10.5.2. LIVE/DEAD™ Fixable Dead Cell Stain Kits (Invitrogen) were used for viability dye staining. For surface staining, splenocytes were stained with the following antibodies: APC anti-mouse CD19 (Clone: 1D3/CD19, BioLegend), PE anti-mouse CD138 (Syndecan-1) (Clone: 281-2, BioLegend), FITC anti-mouse/rat/human CD27 (Clone: LG.3A10, BioLegend), PE anti-mouse CD279 (PD-1) (Clone: 29F.1A1, BioLegend), PE/Cyanine7 anti-mouse CD4 (Clone: GK1.5, BioLegend), and APC/Cyanine7 anti-mouse CD185 (CXCR5) (Clone: L138D7, BioLegend) for Tfh cell analysis; with FITC anti-mouse/human GL7 Antigen (Clone: GL7, BioLegend), PerCP/Cyanine5.5 anti-mouse CD95 (Fas), (Clone: SA367H8, BioLegend), and anti-B220-PerCP-Cy5.5 (Clone: RA3-6B2, BD Pharmingen™) mAb for GC B-cell analysis. Alexa Fluor 700 anti-mouse CD3 (Clone: 17A2, BioLegend), PE anti-CD8a-200 (Clone: 53-6.7, BioLegend), Brilliant Violet 605™ anti-mouse CD69 (Clone: H1.2F3, BioLegend), FITC anti-mouse CD107a (LAMP-1) (Clone: 1D4B, BioLegend).

The tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin and cut into 4 µm sections with HistoCore Autocut (Leica Biosystems, MA, USA). Then, the deparaffinized and rehydrated sections were stained with the primary antibodies anti-CD3ε rabbit mAb (Cell Signaling Technology, MA, USA) at 1:200 dilution, anti-CD8α rabbit mAb at 1:200 dilution (Cell Signaling Technology), and Brilliant Violet 605 anti-mouse CD69 at 1:100 dilution (BioLegend) and labeled with a TSA-Rab multiplex immunofluorescence staining kit (Panovue, Beijing, China) according to the manufacturer’s instructions. The cell nuclei of the sections were stained with DAPI before being visualized by an LSM880.