A11003

KG1A cells arrested with nocodazole (0.1 µg mL⁻¹) for 16 h were treated with indicated concentration of PROTACs, MLN8237 or DMSO for 6 h. Cells were collected, washed and resuspended with PBS and centrifuged onto glass slides. Cells were then fixed with 4% paraformaldehyde (BL539A, Biosharp) for 10 min, permeabilized with 0.1% Triton X‐100 for 5 min and washed with PBS. After blocking with 3% BSA, cells were incubated with the primary antibodies at 4 °C overnight, washed and incubated with secondary antibody at room temperature for 1 h followed by DAPI staining (D9542, Sigma‐Aldrich) and washing step. Finally, the samples were sealed with antifade mounting medium (P0126, Beyotime) and coverslips. The images were taken with laser scanning confocal microscope.
The primary antibodies used were AURKA rabbit mAb (1:200, CST, 14475S) and α‐Tubulin mouse mAb (1:200, AT819, Beyotime). The secondary antibodies used were Alexa Fluor 488 goat antirabbit IgG (H+L) (1:200, A11034, Invitrogen) and Alexa Fluor 546 goat antimouse IgG (H+L) (1:200, A11003, Invitrogen).

MKN45 and MGC803 cells were fixed in 4% paraformaldehyde solution after being transfected with pCDNA3.1-ADAMTS19-3xFlag using a Lipofectamine 3000 reagent and cultured for 48–72 h. The cell membranes were penetrated with a 0.25% Triton X100 (meilunbio, Dalian, China) solution. After 15 min, 1% BSA was transferred to block the cells at room temperature for 30 min. Primary antibodies (Flag-tag: 390002, ZenBio, Sicuan, China, 1:1000; P65: 10745-1-AP, Proteintech, Wuhan, China, 1:200) were incubated at 4 °C for 12–16 h. Primary antibody binding and nuclei were observed using fluorescence secondary antibodies (anti-rabbit: A11034, Invitrogen, 1:200; anti-mouse IgG: A11003, Invitrogen, 1:200) and DAPI (4’,6-diamidino-2-phenylindole; #C1002, Beyotime, Shanghai, China, 1:1000) staining, respectively. Finally, the transfected MKN45 and MGC803 cells were observed and photographed using a confocal microscope (Leica, Weztlar, Germany).

NPM1 (ABclonal #A17983, 1:150) and fibrillarin (monoclonal 38F3, Abcam #ab4566, 1:1000) primary antibodies were used for the overnight incubation at 4°C. Anti-mouse (Invitrogen #A-11003, 1:800) and anti-rabbit (Invitrogen #A-11008, 1:800) secondary antibodies were used for the second incubation on the next morning.

Immunostaining was performed on cultured BMDMs and frozen sections of human and mouse tissues fixed by 2% paraformaldehyde following a previously described protocol.^[18 ,
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^] In brief, sections were labeled overnight with various combinations of directly conjugated primary antibodies as follows: fluorescein isothiocyanate (FITC)‐conjugated anti‐CD68 antibody (sc‐20060 FITC, Santa‐cruz), FITC‐conjugated anti‐F4/80 (11‐4801‐81, eBioscience), Cy3‐conjugated α‐SMA antibody (C6198, Sigma), Runx1 (sc‐365644, Santa Cruz), p‐Runx1(Ser397) (PA5‐105609, Invitrogen) detected with APC (A21240, Invitrogen) or PE‐conjugated secondary antibodies (A11003, Invitrogen). Sections were washed and, in some cases, DNA was counterstained with DAPI and observed under a fluorescence microscope (Carl Zeiss Axio Observer Z1) or confocal microscope (Carl Zeiss LSM 880).

The immunostaining was performed as described (Jasenčáková et al., 2001 (link)). EYFP-FIB1 was detected with primary mouse antisera against FIB1 (1:100; ab4566; Abcam) and secondary antibodies goat anti-mouse-Cy5 (Alexa Fluor^® 647; 1:300; A21235; Invitrogen) or with a goat anti-mouse-Cy3 (Alexa Fluor^® 546; 1:300; A-11003; Invitrogen) for nuclei or metaphase chromosomes, respectively. Alternatively, EYFP-FIB1 on metaphase chromosomes was detected with rabbit antisera against GFP (1,100; ab290; Abcam) recognizing also EYFP and secondary antibodies goat anti-rabbit-Cy3 (Alexa Fluor^® 647; 1:300; A-11010; Invitrogen) for metaphase chromosomes. Nuclei and chromosomes were counterstained with DAPI dihydrochloride (1 μg mL⁻¹) in a Vectashield medium (Vector Laboratories).
Microscopic images were acquired using a Leica TCS SP8 STED3X confocal microscope (Leica Microsystems, Wetzlar, Germany), equipped with an HC PL APO CS2 63 ×/1.40 Oil objective, hybrid detectors, and the LAS-X software version 3.5.5 with the Leica Lightning module (Leica, Buffalo Grove, IL, United States). Confocal images were captured separately in sequential scans, to avoid spectral mixing, using 405 nm (DAPI), 508 nm (EYFP), 557 nm (Alexa Fluor^® 546), and 594 nm (Alexa Fluor^® 647) laser lines for excitation and appropriate emission spectrum. Pictures were processed in Adobe Photoshop version 12.0 (Adobe Systems).

Coronal sections (80 μm) were obtained with a vibratome (Leica, Germany), permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) and incubated with 3% bovine serum albumin (Sigma-Aldrich). After standard antigenic retrieval using sodium citrate, the free-floating sections were incubated overnight with the following primary antibodies: anti-Caspase-3 (1:300, Millipore, AB3623); rabbit anti-Ki67 (1:100, Millipore, AB9260); mouse anti-NS1 (1:10, Thermo Fisher Scientific, MA5-24585); rabbit anti-SOX2 (1:200, Sigma-Aldrich/Millipore, AB5603); rabbit anti-SOX9 (1:200, Abcam, ab185230); mouse anti-IBA1 (1:1000, Millipore, MABN92); mouse anti-NEUN (1:200, Millipore, MABN377); and rabbit anti-OLIG2 (1:300, Millipore, MABN50). Following the overnight incubation, sections were washed with PBS and incubated for 2 h with goat anti-rabbit Alexa Fluor 488 (1:500, Millipore, AP132JA4) and goat anti-mouse Alexa Fluor 546 (1:500, Invitrogen, A11003) secondary antibodies. The free-floating sections that were stained with isolectin B4 (IB4; 1:200, Invitrogen, 121411) were incubated with the stain together with the secondary antibodies. Nuclei were stained with DAPI (0.5 μg/ml) for 30 min. Sections were placed in glass slides and mounted with Fluoromount (Sigma-Aldrich, F4680).