Ab202073

B cells isolated from human PBMCs by Magnetic-Activated Cell Sorting (17954, StemCell Technologies) were immobilized onto glass slides coated with poly-L-lysine (Sigma-Aldrich). The BCR was labeled with a human IgM+G antibody (Alexa Fluro 546-(Fab)2-anti-Ig(M+G)). This antibody was generated from the F(ab′)2 fragment (Jackson ImmunoResearch, West Grove, PA) using a published protocol (23 (link)). Alexa Fluro 546-(Fab)2-anti-Ig(M+G) was incubated with B cells for 30 min on ice before Fc receptor blocking (422301; BioLegend). Then, the B cells were stimulated for 0, 5, 15, or 30 min at 37°C before fixation with 4% paraformaldehyde. In some experiments, B cells were stimulated for 48 h at 37°C with CpG (10 μg/mL) and CD40L (1 μg/mL) to detect activation of phospho-STAT3. The following specific antibodies were used to stain the cells after permeabilization with 0.05% saponin (Sigma): anti-CD38 (ab235118; abcam), anti-CD24 (ab202073; abcam), anti-phospho-STAT3 (9145S; Cell Signaling Technology), anti-phosphotyrosine (05-1050X; Merck Millipore), anti-pBTK (ab52192; abcam), and anti-pCD19 (3571S; Cell Signaling Technology). Images were obtained under a confocal microscope (Nikon A1R) using 405, 488, 546, 647 nm lasers. Colocalization and MFI were determined by the NIS-Elements AR 3.2 software.

Lens capsule flat mounts or lens sections were fixed with 4% paraformaldehyde for 20 min, washed with phosphate-buffered saline (PBS) three times, and permeabilised with 0.3% Triton X-100 for 40 min. Tissues were blocked in 5% sheep serum and 1% bovine serum albumin dissolved in PBS for 1 h and incubated overnight with primary antibodies at 4 °C. The primary antibodies were used at the following dilutions: SQSTM1/p62 (ab56416, Abcam, Cambridge, UK) at 1:200, CD24 (ab202073, Abcam) at 1:200, CD63 (ab271286, Abcam) at 1:200 and CD81 (ab219209, Abcam) at 1:200. Following three washes in PBS, the tissues were incubated with secondary antibody (Alexa Fluor 488 conjugated anti-rabbit IgG, ab150077, Abcam) at 1:800 for 2 h at room temperature. Cell nuclei were stained with DAPI. Images were obtained using a Zeiss LSM700 confocal microscope.

The CD44 and CD24 expression levels in breast cancer tissues were analysed by immunofluorescence. Briefly, tissue sections (4 µm) were dewaxed, rehydrated, and subjected to antigen retrieval, after which they were treated with 10% FBS and incubated with a mouse anti‐human CD44 monoclonal antibody (#3570; Cell Signaling Technology, Danvers, MA, USA) and a rabbit anti‐human CD24 monoclonal antibody (ab202073; Abcam, Cambridge, UK) at 4ºC overnight. After washing, the sections were incubated with the Alexa Fluor 647‐labelled goat‐anti‐mouse IgG and Alexa Fluor 488‐labelled goat‐anti‐rabbit IgG, followed by nuclear counterstaining with DAPI (C0060; Solarbio, Beijing, China). Fluorescent signals were observed under a fluorescent microscope and the percentages of CD44⁺/CD24^‐ CSCs in 2000 total tumour cells from at least three sections were counted in a blinded manner.