Fitc conjugated anti rabbit secondary antibody

Differentiated THP-1 cells were infected with M. tuberculosis H37Rv and M. tuberculosis H37Rv expressing GFP and were incubated for 4 h to facilitate phagocytosis. Cells were then washed four times with complete RPMI containing gentamicin (10 μg/ml) to remove extracellular bacilli, and fresh medium was added to the cells. Cells were fixed with paraformaldehyde (4%), incubated with methanol for 20 min at −20°C, and then blocked with phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) (3%, wt/vol), and Triton X-100 (0.3%, vol/vol). The cells were incubated for 1 h at room temperature with anti-rabbit or anti-mouse primary antibodies against pHDAC1 (EMD Millipore, USA), ZBTB25 (Sigma-Aldrich, USA), LC3 (Santa Cruz Biotechnology, USA), ATG5 (Santa Cruz Biotechnology, USA), and BECN1 (Santa Cruz Biotechnology, USA), washed, and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody (Sigma-Aldrich, USA) or Alexa 488/Alexa 592 anti-rabbit or anti-mouse secondary antibody (Thermo Fisher, USA) for 1 h at room temperature. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for DNA. To detect lysosomes, cells were incubated with LysoTracker Red (Thermo Fisher Scientific, USA). Rapamycin (Sigma-Aldrich, USA) was used as a positive control for autophagy induction.

RS4;11 and SEM smears were fixed using a cytology fixative (Bio-FIX 05-X200, Bio-Optica, Italy). SKBR3 were treated with 15 μM of JSH-23 NF-κB inhibitor (ab144824, abcam) for 3 h at 37 °C. Subsequently, cells were fixed using freeze (− 80 °C) methanol for 10 min. The slides were subjected to blocking with a solution of 3% (w/v) BSA in PBS pH 7.4 at Room Temperature. Anti NF-κB (Rabbit, 622602 Biolegend, USA) antibody was diluted 1:100 in a solution of PBS + 1% (w/v) BSA and then all slides were incubated for 4 h at + 4 °C. After five wash steps in PBS for 5 min each, FITC-conjugated anti-rabbit secondary antibody (F0382, Sigma Aldrich) diluted 1:200 in a solution of PBS + 1% (w/v) BSA was incubated for 1 h at 4 °C in the dark. For SKBR3 an anti β-tubulin (1:100) followed by a PE- conjugated anti-mouse secondary antibody (1:200, A10543, Thermo Fischer) were added to detect cytoplasm. After an additional five wash steps in PBS, a solution of 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fischer Scientific D1306) diluted 1:35,000 in PBS was used for coloring of the nuclei.
Images were obtained using an automated upright microscope system (Leica DM5500 B) coupled with Leica Cytovision software.

Immunofluorescent staining was performed as described in a previous study¹⁸ . Briefly, DLBCL (2 × 10^{6 (link)}) cells were cultured in 6-well plates. After cytospinning at 350 rpm for 15 min, cells were transferred onto poly-L-lysine-coated glass slides for immunofluorescence staining. Formaldehyde (100 μl, 4%) was added to fix cells at room temperature for 15 min followed by 0.1% triton for 15 min. After washing with PBS, 3 drops of background-reducing reagent (Dako, S3022, Carpinteria, CA, USA) were added. The cells were incubated with 50–100 μl primary antibodies at 4 °C overnight and then incubated at room temperature in the dark for 1 h with FITC-conjugated anti-rabbit secondary antibody (1:150; Sigma-Aldrich, St. Louis, MO, USA). Nuclear DNA was stained with 4′-6-diamidino-2-phenylindole (DAPI; 1:1000; Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature in the dark. Finally, the cell signal was detected by fluorescence microscopy. The used primary antibodies included NANOG (polyclonal, 1:20, ab21624, Abcam), and HOXA9 (polyclonal, 1:20, ab92565, Abcam).