Alexa fluor 594

Secondary antibodies used were: Donkey αMouse, Alexa Fluor 594 (Dianova, 711585150); Donkey αRabbit, Alexa Fluor 488 (Invitrogen, A21206); Goat αMouse, Alexa Fluor 488 (Invitrogen, A11001); Donkey αGoat, Alexa Fluor 488 (Invitrogen, A11055); Donkey αGoat, Alexa Fluor 555 (Invitrogen, A21432).

Commercial antibodies were obtained from following companies: Abnova (Taipei City, Taiwan): UBTF (H00007343-M01), Cell Signaling (Danvers, MA, USA): GAPDH (1410C), p21 (2946); Proteintech (Rosemont, CA, USA): PPAN (11006-1-AP), ATG7 (67341-1-Ig) and SBDS (67200-1-Ig); Sigma (Steinheim, Germany): PES1 (SAB1400457); Thermo Fisher Scientific (Dreieich, Germany): NPM (FC-61991); Roche (Mannheim, Germany): GFP (11814460001); Santa Cruz (Dallas, TX, USA): p53 (sc-126). Secondary antibodies for Western blotting were IRDye conjugates 800CW and 680CW from Li-COR (Lincoln, NE, USA) or Alexa Fluor 594 (Dianova, Hamburg, Germany) for immunofluorescence experiments. DAPI mounting medium were purchased from Dianova (Hamburg, Germany).

For indirect immunofluorescence microscopy analyses, BMM were cultured on 10 mm coverslips in 24‐well dishes. At indicated points of time, cells were washed three times with equilibrated PBS, fixed for 15 min with equilibrated 4% paraformaldehyde (PFA) and permeabilized with ice‐cold methanol for 1 min. Cells were quenched and blocked with 50 mM NH₄Cl in PBS/5% goat serum (GS) for 60 min at room temperature. Incubation with primary antibody dilution in PBS/5% GS was conducted at room temperature for 60 min. Subsequently, cells were washed three times with PBS and further incubated with secondary antibodies in PBS/5% GS for 30 min at room temperature. After final 3× washing with PBS, coverslips were mounted using ProLong Diamond containing DAPI. For visualization, a Carl Zeiss LSM 700 Laser Scan Confocal Microscope and the ZEN2009 software (Jena, Germany) were used.
In this study, we used primary antibodies directed against C. burnetii (polyclonal rabbit serum, Davids Biotechnologie, Regensburg, Germany) as a 1:5,000 dilution and LAMP‐1 (monoclonal rat IgG, Developmental Studies Hybridoma Bank, Iowa, IA, USA) as a 1:400 dilution. Secondary antibodies were labeled with Alexa Fluor 594 and Alexa Fluor 488, respectively (both were used as 1:800 dilution) (Dianova, Hamburg, Germany).