Tritc conjugated goat anti rabbit igg

Diverse primary antibodies were used: rabbit anti-Iba1 (IgG; 1:100), a marker of quiescent and active microglia (Waco Chemicals, Osaka, Japan), mouse anti-glial fibrillary acidic protein (GFAP) (IgG2b; 1:100) (BD Pharmingen Franklin Lakes, NJ, USA), mouse anti-neuN (IgG1; 1:100), mouse anti-rat CD68/ED1 (IgG1; 1:100), a marker of activated microglial cells in a phagocytic state (Serotec, Oxford, UK). Secondary antibodies were used at 1:100 dilution: FITC and Tetramethyl Rhodamine IsoThioCyanate (TRITC)-conjugated goat anti-mouse IgG (γ-chain specific and against whole molecule respectively), TRITC-conjugated goat anti-rabbit IgG (whole molecule), FITC-conjugated sheep anti-rabbit IgG (whole molecule) (Sigma, Saint-Louis, MO, USA), FITC and TRITC-conjugated donkey anti-mouse IgG (whole molecule), FITC and TRITC-conjugated donkey anti-sheep IgG (whole molecule), Cy3-conjugated donkey anti-rabbit IgG (whole molecule) and anti-goat IgG (whole molecule) (Jackson ImmunoResearch Laboratories, Inc., WestGrove, PA, USA).

After washing with PBS, cells plated on cover slips were fixed with 4% paraformaldehyde for 30 min. Cells were then permeabilized with 0.1% Triton X-100 for 5 min. After being blocked with complete serum for 30 min at 37°C, the cells were incubated with primary antibody at 4°C overnight. The cells were then incubated with FITC-conjugated goat anti-mouse IgM or TRITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Images were captured with an Olympus BX83 fluorescence microscope (Japan).

The day before use, THP-1 cells were seeded onto glass coverslips coated with poly-L-lysine, which were then placed in 24-well plates (1 × 10⁵ cells/well). Cells were incubated with 5 μM each of the 3D8 antibodies for 6 h at 37°C, fixed for 10 min at RT in 4% paraformaldehyde (PFA) in PBS, and permeabilized by incubating for 10 min at RT with Perm-buffer (1% BSA, 0.1% saponin, and 0.1% sodium azide in PBS). Cells were then incubated for 1 h at 4°C with Dylight 550-conjugated goat anti-human IgG/Fc (Abcam, Cambridge, UK; cat# 97004) to detect wt 3D8 IgG, 3D8 IgG-N434D, 3D8 scFv-Fc, and polyclonal human IgGs. To detect 3D8 scFv, cells were incubated for 1 h at 4°C with a primary antibody (polyclonal rabbit anti-3D8 scFv), followed by TRITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich; cat# T6778). Each incubation step was followed by three washes with cold PBS (pH 7.2). Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific; cat# 62249) for the last 10 min of incubation at RT. Cells (on coverslips) were mounted in Vectashield anti-fade mounting medium (Vector Labs, Burlingame, CA, USA) and observed under a Zeiss LSM 710 laser confocal microscope.

After washing with PBS, cells grown on coverslips were fixed with 4% paraformaldehyde for 30 min. Cells were then permeabilized with 0.1% Triton X-100 for 5 min. After being blocked with 3% serum for 30 min at 37 °C, cells were incubated with rabbit anti-HDAC3 antibody (1∶200) or Ac-p53 (k373/k382) (1∶200) at 4 °C overnight. The cells were then incubated with FITC or TRITC-conjugated goat anti-rabbit IgG (1∶200) (Sigma-Aldrich, St. Louis, MO) for 1 h. Images were captured with the Olympus BX83 fluorescence microscope (Japan).