Dm500b

The optimized antibody E15, which only recognizes CARM1FL, and E16, which recognizes both isoforms, were generated and specificity was verified using blocking peptides as previously described [19 ]. Immunofluorescence: baseline levels of autofluorescence from the secondary antibody were determined by IF using secondary antibody only. Cells were fixed in 4% paraformaldehyde in PBS then blocked in 3% BSA in PBST for twenty minutes, followed by a one hour incubation in primary antibody E15 or E16 (1:300) diluted in 3% BSA, or 3% BSA alone (control). After washing with PBS + 0.1% Triton (PBST), cells were incubated for one hour in secondary antibody goat-anti-rabbit conjugated to FITC (1:1000) diluted in PBST. 50 μL of phalloidin-Alexa 555 were added to each coverslip for fifteen minutes, and then slides were washed with PBST. Fifteen μL of DAPI with Prolong Gold were added to each slide and slides were visualized by fluorescence using a wide-field microscope (LEICA DM500B) with immersion oil on a 100x/1.30 numeric aperture lens. Exposure time, saturation, gamma, gain, and other camera settings were kept constant for images of each fluorophore for each cell line. Fluorescence intensity was measured using Nikon AR Elements software Western blotting of CARM1 was performed as described previously [6 (link)].

The nuclear morphology of the hemocyanin-treated cells was examined by fluorescent microscopy after staining with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) according to the manufacturer instructions. Graffi tumor cells were seeded and cultured with and without the hemocyanins, as described for the AO/EB staining. After 24 h of treatment, the glass lamellas with adherent tumor cells were washed, fixed with methanol, and stained for 15 min with 1 µg/mL DAPI in methanol at room temperature in the dark. Stained cells were mounted with glycerol on microscope slides and analyzed by a fluorescent microscope (Leica DM 500B, Wetzlar, Germany).

Parts of spinal cords were immersed in 4% paraformaldehyde for 8 h and 30% sucrose overnight at 4°C. After embedded and sliced, the sections were incubated with 0.3% Triton X-100 for 20 min, 5% donkey serum for an hour, and primary antibody in 1% donkey serum overnight at 4°C. Rabbit anti-NF-κB(p65) antibody (Cell Signaling Technology, USA, 1:200) and goat anti-GFAP antibody(Abcam, UK, 1:200) were used. After being washed again, sections were respectively treated with donkey anti-rabbit IgG Dylight488 and goat IgG Dylight594 (1:200, Jackson ImmunoResearch Laboratories, USA). Then, the sections for NF-κB(p65) detection were treated with DAPI for 10 min. Then, the sections were washed with phosphate-buffered saline for three times. All sections were visualized with fluorescence microscope (Leica DM500B, Wetzlar, Germany).