A 21050

Immunohistochemistry was carried out as previously described (Kroehne et al., 2011 (link)). Briefly, for detection of HuC/D, sections were subjected to antigen retrieval by 15 min incubation in 10 mM citrate buffer (pH 6.8). Primary and secondary antibodies were incubated in PBS with 0.3% Triton X-100 (PBS TX). Tissue sections were incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature. The slides were then washed in PBS TX and mounted. We used primary antibodies to HuC/D (Elavl3) (mouse, Thermo Fisher Scientific, A21271, 1/250). For primary antibody validation, see www.thermofisher.com/antibody/product/HuC-HuD-Antibody-clone-16A11-Monoclonal/A-21271. Alexa Fluor 633-conjugated secondary antibodies were used (Thermo Fisher Scientific, A-21050, A21422; 1/750). EdU was detected with the EdU Alexafluor 488 Plus Imaging Kit (Thermo Fisher Scientific, C10337) according to the manufacturer's instructions.

Synchronized populations of C. elegans eggs, larvae or adults were freeze-cracked, fixed with -20°C cold methanol for 2 min and cold acetone for 4 min. Next, samples were dried at RT and recovered by adding a drop of PBS containing 0.1% Tween (PBST) for 5 min. Once eggs or worms were properly prepared, they were blocked for 15 to 30 min in 1% BSA PBST blocking solution. Subsequently, samples were incubated either O/N at 4°C or at RT using FLAG antibody in 1% BSA PBST (F1804 Sigma, 1:500 dilution) followed by 1 or 2 hours incubation with the secondary antibody Alexa Fluor 633 in 1% BSA PBST (Invitrogen A-21050, 1:500 dilution). Finally, samples were mounted using VECTASHIELD Antifade Mounting Medium (H-1000 Vector laboratories) with DAPI (1 μg/ml).

Hippocampal cells cultured on glass coverslips were washed with serum free Neurobasal medium (10 mM HEPES), and treated with 1 μM CAM2(Ax488) in serum-free Neurobasal medium (10 mM HEPES) at 17 °C for 4 h. Then, the cells after labelling were washed with HBS buffer and fixed with 4% paraformaldehyde at room temperature (r.t.) for 30 min and washed with PBS buffer. This was followed by permeabilization with PBS containing 0.2% triton X-100 at r.t. for 10 min and blocking with PBS containing 10% normal goat serum for 1 h. After blocking, primary antibody in PBS buffer containing 5% normal goat serum was added and incubated at 4 °C for 12 h. Secondary antibody in PBS buffer containing 5% normal goat serum and was added and incubated at r.t. for 1 h. Used primary antibodies were as follows: mouse anti-GluA2 (Millipore, MAB397, × 300), mouse anti-PSD95 (abcam, ab2727, × 300) and rabbit anti-MAP2 (Millipore, AB5622, × 300). Secondary antibodies were conjugated to Alexa546 or Alexa633 fluorophores (Invitrogen, A11071, A11018, A21050, or A21070, × 1,000). Cell imaging was performed with a confocal microscopy (LSM710, Axio Observer.Z1, ZEISS) equipped with a 63 × , numerical aperture (NA)=1.40 oil objective. Fluorescence images were acquired using a 488 nm line of an argon laser for excitation of Ax488, DPPS laser for excitation of Alexa546 and HeNe laser for excitation of Alexa633.

The materials used in this study are as follows: mouse mononuclear macrophage leukemia cell line (Raw264.7, Chinese Academy of Sciences, China), Roswell Park Memorial Institute (RPMI) 1640 cell culture medium (Thermo Fisher, USA), HDAC9-shRNA and NC-shRNA (Obio Technology Corporation, Ltd. Shanghai, China), phosphate buffer saline pH 7.4 (Sigma Aldrich, USA), Triton X-100 (T8200, Soleibo, China), TLR4 inhibitor (B4935, APExBio Shanghai, China), HDAC9 (PA5–11245, 1 : 1000) and TLR4 (MAI-33380, 1 : 1000) (Invitrogen, USA, 1 : 1000), IL-1β (A1112, ABclonal, China, 1 : 500), CD38 (A20215, ABclonal, China, 1 : 500), TNF-α (A11534, ABclonal, China, 1 : 500), GAPDH (WL01114, Wanleibio, China, 1 : 10000), HRP goat anti-rabbit IgG (AS014, ABclonal, China, 1 : 10000), and fluorescent secondary antibodies A-11008 and A-21050 (Invitrogen, USA). All chemicals were used without any further processing.

For immunofluorescence assay, AML12 cells with indicated treatments were cultured in confocal dish (35 mm) and incubated for 48 h. Then, the culture medium was discarded and the cells were washed with PBS, followed by fix with 4% paraformaldehyde for 20 min. Then, cells were stained with primary antibody (anti-HRS, sc-271455) overnight, followed by secondary antibody [Goat anti-mice 633, Invitrogen, A-21050)] at room temperature for 1 h in the dark. Finally, the nuclei were stained with Hoechst (1:1000). Images were processed using laser scanning confocal microscope. HRS spots per cell were calculated using Image J.

Images were taken with a Nikon Swept Field Confocal (Nikon). For each condition, at least 25 independent visual fields were counted. Mesothelioma cells were washed with PBS, fixed in 4% formaldehyde for 10 min and washed with PBS. Then, the cells were permeabilized for 10 min with 0.1% Triton X-100 in PBS and blocked in PBS containing 2% BSA and 0.05% Triton X-100 for 1 h. The cells were then incubated with primary antibody (anti-cytochrome c and anti-HSPD1) for 3 h at room temperature and washed three times with PBS. The appropriate isotype-matched, AlexaFluor-conjugated secondary antibodies [Life Technologies, A11008 (488 goat anti-rabbit) and A-21050 (633 goat anti-mouse)] were used. Images were obtained with a Nikon Swept Field Confocal equipped with a CFI Plan Apo VC60XH objective (n.a. 1.4) (Nikon Instruments, Melville, NY) and an Andor DU885 EM-CCD camera (Andor Technology Ltd, Belfast, Northern Ireland). Statistical evaluation was performed using the colocalization counter JACOP, available in the Fiji software.