A 21424

Rabbit anti-human SAP polyclonal antibody (pAb) (565191, Calbiochem), rabbit anti-human CRP pAb (235752, Calbiochem), rabbit anti-human C1q pAb (A0136, Dako), mouse anti-human complement component C5b-9 monoclonal antibody (mAb) (IgG2a) (011-01, Antibody Shop), mouse IgG1 isotype control (BD Biosciences), mouse IgG2a isotype control (BD Biosciences), rabbit IgG isotype control (Invitrogen), FITC-conjugated goat anti-rabbit pAb (F1262, Sigma-Aldrich), FITC-conjugated goat anti-mouse pAb (F0479, Dako), mouse anti-human CD45 FITC/CD14 PE (342408, BD Biosciences), mouse anti-human CD11b APC-Cy7 (560914, BD Biosciences), Alexa-555-conjugated goat anti-mouse IgG (A-21424, Thermo Fischer), biotinylated goat anti-rabbit IgG (BA-1000, Vector Laboratories), or biotinylated goat anti-mouse IgG (BA-9200, Vector Laboratories) are the commercial antibodies.

Wild-type H4 cells were plated at 70–80% confluency in 24-well plates containing poly-D lysine treated glass cover slips. For knockdown experiments, cells were transfected with siRNAs either targeting BAG5 (siBAG5, Thermo Fisher Scientific 4392420) or non-targeting control (siNTC, Thermo Fisher Scientific 4390843) at the time of plating using Lipofectamine RNAiMAX (Thermo Fisher Scientific) as per the manufacturer’s protocol. 48 h after transfection, cells were washed once with PBS and treated with 4% paraformaldehyde (PFA) for 15 min at room temperature. The PFA was washed off with three sequential 5 min PBS washes and the cells were subsequently treated with 0.2% triton X-100 diluted in PBS for 15 min at room temperature. Cells were then washed another three times in PBS and blocked with 5% weight/volume bovine serum albumin (BSA) diluted in PBS for 45 min. BAG5 and p62 antibodies were diluted 1:1000 in 5% BSA/PBS and incubated with the cells overnight at 4°C with gentle rocking. Cells were washed in PBS, incubated in species specific Alexa Fluor 488 and 555 (Thermo Fisher Scientific A11034 and A21424, respectively), and washed again in PBS containing 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Finally, the coverslips were mounted onto slides with fluorescent mounting media (Dako, S3023).

Whole eyes were cryo-preserved by immersion in 30% sucrose overnight at 4 °C, followed by embedding in optimal cutting temperature compound (OCT) (Sakura Finetek). Eyes were then frozen on dry ice and 13 μm sections collected through the dorsal–ventral/superior–inferior axis of the retina onto super frost plus slides using a OTF5000 cryostat (Bright instruments). Sections were washed in PBS and blocked in 5% normal goat serum (NGS) (G2023; Sigma-Aldrich), 2% BSA, and 0.3% Triton X-100 in PBS for 60 min at room temperature. Sections were then incubated in primary antibodies against RBPMS (1832; 1:500; PhosphoSolutions), Prox1 (925202; 1:500; BioLegend), Calretinin (ab702; 1:500; Abcam), Calbindin (ab11426; 1:500; Abcam), PKC-α (sc-8393; 1:500; Santa Cruz Biotechnology) or Vimentin (ab5733; 1:500; Millipore). Afterwards, the tissue was washed in PBS then incubated with secondary antibodies anti-guinea pig AF 555 (A21435; 1:1000; Thermo Fisher Scientific), anti-chicken AF 568 (A11041; 1:1000; Thermo Fisher Scientific), anti-rabbit AF 647 (A32733; 1:1000; Thermo Fisher Scientific) or anti-mouse AF 555 (A21424; 1:1000; Thermo Fisher Scientific) with DAPI (D1306; 1:8000; Thermo Fisher Scientific) in the above-mentioned blocking detergent for 2 h at room temperature. The retinal sections were washed in PBS and then mounted using FluorSave^TM reagent.

SW1353 cells were transfected with Flag-Zfhx4, Venus-Osterix, or Venus-Runx2 using the X-tremeGENE 9. After 48 h, cells were washed twice with PBS and fixed with 4% buffered paraformaldehyde (WAKO, Osaka, Japan) for 20 min. After treatment with 0.2% Triton X-100 in PBS for 5 min, cells were blocked with 1% BSA in PBS for 1 h, incubated with an anti-Flag (WAKO) antibody at room temperature for 2 h, and then incubated with Alexa Fluor 555-conjugated anti-mouse IgG (1:500, A21424; Thermo Fisher Scientific) for 30 min. Nuclear staining was performed using Vectashield with 4′,6-diamidino-2-phenylindole (Vector, Burlingame, CA, USA). Samples were visualized using a Leica TCS SP8 confocal microscope (Leica Microsystems).

Cells were treated with 10 nM trabectedin (MedChemExpress) for 4 h. During the last 15 min, 20 µM 5-ethynyl-2′-deoxyuridine (5-EdU; Jena Bioscience) was added. Cells were then washed with 300 mM sucrose (Merck) in PBS on ice, pre-extracted with 300 mM sucrose and 0.25% Triton X-100 in PBS for 2 min on ice and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature. Cells were then permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature and blocked in 3% BSA (Thermo Fisher Scientific) in PBS. Dividing cells were visualized by click-it chemistry, labeling the cells for 30 min with a mix of 60 µM Atto azide-Alexa 594 or Atto azide-Alexa 647 (Atto Tec), 4 mM copper sulfate (Sigma-Aldrich) and 10 mM ascorbic acid (Sigma-Aldrich) in a 50 mM Tris buffer. After washing with PBS, cells were blocked with 100 mM glycine (Sigma-Aldrich) in PBS for 10 min at room temperature and subsequently with 0.5% BSA and 0.05% Tween 20 in PBS for 10 min at room temperature. To visualize γH2AX, cells were incubated with a primary antibody for phospho-Histone H2A.X Ser139 (JBW301, Merck) for 2 h at room temperature and then with a secondary antibody, anti-mouse Alexa 555 (A-21424, Thermo Fisher Scientific) or anti-mouse Alexa 647 (A-21235, Thermo Fisher Scientific), and DAPI for 1 h at room temperature and mounted in Polymount (Brunschwig).