Anti npm1 fc 61991

For immunofluorescence microscopy, cells were cytospinned on glass slides and subsequently fixed using 4% paraformaldehyde in PBS. Subsequently, cells were permeabilized using PBS containing 0.1% Triton X-100. Primary antibodies include anti-Fibrillarin (ab5821, Abcam), anti-NPM1 (FC-61991, Thermo Fisher Scientific), anti-CBX4 (E6L7X, Cell Signalling Technology) and anti-EZH2 (D2C9, Cell Signalling Technology) followed by secondary antibody staining using Alexa Fluor 488 goat-anti-rabbit (Thermo Fisher Scientific, A-11008), Alexa Fluor 488 goat-anti-mouse (Thermo Fisher Scientific, A-11001), Alexa Fluor 647 goat-anti-rabbit (Thermo Fisher Scientific, A-21244), or Alexa Fluor 647 goat-anti-mouse (Thermo Fisher Scientific, A-21235). Images were acquired on a Leica DM6000B microscope using a 40x dry objective (HCX PL FLUOTAR, numerical aperture: 0.75) or a 63x immersion objective (PL S-APO, numerical aperture: 1.30) using LAS-AF software (Leica). Confocal images were acquired on a Leica TCS SP8 confocal laser-scanning microscope using a HC PL APO CS2 63x/1.4 oil objective, and excitation with 488 nm (20 mW) and 633 nm (30 mW) laser lines.

Tissues were embedded in Tissue‐Tek OCT medium and snap frozen in liquid nitrogen. For immunofluorescence, 10‐μm cryosections were rehydrated to water, postfixed with 4% PFA/PBS (Sigma‐Aldrich), blocked and permeabilized for 30 min in .25% Triton X‐100/5% normal donkey serum (NDS)/PBS (Vector Laboratories). Antibodies were diluted in 5% NDS/.1% Tween‐20/PBS. The following primary antibodies were incubated at 4°C overnight: anti‐FAPα (AF3715, R&D Systems), anti‐CXCL12 (MA5‐23759, Thermo Fisher Scientific), anti‐THBD (hpa002982, Atlas Antibodies), anti‐NPM1 (FC61991, Thermo Fisher Scientific), anti‐FBLN1(hpa001612, Atlas Antibodies), anti‐CD90 (AF2067, R&D Systems), anti‐c‐Myc (MAB36961, R&D Systems) and anti‐c‐Fos (PC05L, Merck). Staining was visualized with secondary antibodies (IgG‐NL493 (NL006) and IgG‐NL557 (NL010), 1:200, R&D Systems). Nuclei were visualized using ProLong Gold Antifade Mountant (P36931, Thermo Fisher Scientific). Stains were evaluated with a Leica DM6000 fluorescence microscope and using LAS AF Lite software.