Anti rabbit igg fab2 alexa fluor 488

Prior to ICC, coverslips needed to be put at the bottom of the wells in a 24-well plate. In total, 1 × 10⁵ HER2-positive breast cancer cells (SK-BR3, MDA-MB361, and JIMT1) and HER2-negative breast cancer cells (MCF7) were seeded on the coverslips. Cells were fixed in 4% paraformaldehyde for 15 min and washed with phosphate-buffered saline (PBS) twice. Nonspecific sites were then blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich, Burlington, MA, USA) in PBS for 30 min at room temperature. Cells were incubated with anti-HER2 antibody (HER2/ErbB2 (D8F12) XP TM rabbit mAb, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C and washed with PBS, followed by incubation with the appropriate Alexa Fluor 488 secondary antibody (1:1000, anti-rabbit IgG Fab2, Alexa Fluor ^® 488, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The coverslips were added to the mounting medium with DAPI (Vectashield Mounting Medium with DAPI, Tokyo, Japan) for 10 min and were observed using a fluorescence microscope (LSM 700 confocal, ZEISS, Oberkochen, Germany).

NPM1 construct GFP-NPM1 WT (plasmid ID: 17578) was purchased from Addgene Inc. (Cambridge, MA, USA), and then subcloned into the pET28a vector to express the fusion protein with an N-terminal 6X histidines and T7 epitope tags. The recombinant protein expressed in Escherichia coli BL21 (DE3) was purified using nickel column chromatography. Polyclonal anti-NPM1 rabbit antibody and monoclonal anti-β-actin mouse antibody were obtained from commercial sources (Cell Signaling Technology, Inc., Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-human IgG, HRP-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-human IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-rabbit IgG Fab2 (Alexa Fluor 488) was purchased from Cell Signaling Technology, Inc.