Alexa fluor 647

To detect bat lyssavirus antigen in infected brains, a polyclonal rabbit serum against recombinant RABV P protein (P160-5, 1:3,000 in PTwH [0.2% Tween 20 in PBS with 10 μg/ml heparin]) was used [50 (link)]. The following commercial primary antibodies were used: chicken anti-GFAP (Thermo Fisher, USA; #PA1-1004, RRID:AB_1074620, 1:1,500 in PTwH) and guinea pig anti-NeuN (Synaptic Systems, Germany; #266004, RRID:AB_2619988, 1:800 in PTwH). Donkey anti-rabbit Alexa Fluor 568 (Thermo Fisher, USA; #A10042, RRID:AB_2534017) and donkey anti-guinea pig Alexa Fluor 647 (Dianova, Germany; #706-605-148, RRID:AB_2340476) were used as secondary antibodies, each at a dilution of 1:500 in PTwH.

Antibodies used for immunofluorescence were: gammaH2AX (Millipore, Darmstadt, Germany, 05-636) 1:400, 53BP1 (Santa Cruz, Dallas, TX, USA, sc-22760) 1:500, KAP1 (Abcam, Cambridge, UK, ab22553) 1:400, pKAP1 (Abcam ab133440) 1:200, H3K9me3 (Abcam ab8898) 1:500, H4K8ac (kind gift from Dr. H. Kimura, Osaka University), CAR (Millipore ab15282), 1:500, lamin B (Santa Cruz sc-6217) 1:400, lamin A/C (Millipore #05-714), LBR (Biozol, Eching, Germany, bs-5081R) 1:200, 1:500, Alexa Fluor488 (Invitrogen A11001, A11008) 1:400, Alexa Fluor594 (Invitrogen A11005, A110012) 1:400, Alexa Fluor647 (Dianova, Hamburg, Germany, 715-605-150) 1:400, Dylight 550 (Dianova, A-24421-05).

HepaRG and HepG2 cells were grown on cover slides for indirect immunofluorescence. At different times posttransfection or infection, the samples were fixed by incubation in 4% paraformaldehyde (PFA) for 10 min at room temperature. For HBV-infected HepG2 cells, an additional CSK washout was performed in indicated samples as recently described (42 (link)). Prior to blocking in Tris-buffered saline–BG (TBS-BG; BG is 5% [wt/vol] bovine serum albumin [BSA] and 5% [wt/vol] glycine) buffer for 30 min at room temperature, the samples were permeabilized in 0.5% Triton X-100 for 5 min. The cover slides were incubated with primary antibody diluted in phosphate-buffered saline (PBS) overnight at 4°C. Samples were washed three times with TBS-BG and incubated in the corresponding Alexa Fluor 488 (Invitrogen)- or Alexa Fluor 647 (Dianova)-coupled secondary antibodies. Prior to mounting in Mowiol, the slides were washed three times in TBS-BG. Samples were analyzed using a confocal laser-scanning microscope (Nikon TiE) and the Volocity software, a Zeiss LSM 980 laser scanning microscope and the Zeiss Zen 3 software, or a Zeiss Axio Observer Z.1 microscope and the Axiovision 4.8 software. To quantify colocalization, Spearman correlation ranks of the respective fluorescence channels were determined using Fiji (76 (link)) and the Pearson-Spearman correlation colocalization plugin (77 (link)).

To detect RABV in the tissues, a polyclonal rabbit serum against recombinant RABV P protein (P160-5; 1:3000 in PTwH [0.2% Tween 20 in PBS with 10 µg/mL heparin]) was used [45 (link)]. The following first antibodies and dyes were obtained from the respective suppliers: chicken anti-NEFM (Thermo Fisher, USA; #PA1-16758, RRID:AB_2282551; 1:1000 in PTwH), guinea pig anti-NEFM (Synaptic Systems, Germany; #171204, RRID:AB_2619872; 1:400 in PTwH), chicken anti-MBP (Thermo Fisher; #PA1-10008, RRID:AB_1077024; 1:500 in PTwH), TO-PRO™-3 iodide (Thermo Fisher; #T3605; 1:1000 in PTwH.) As secondary antibodies donkey anti-rabbit Alexa Fluor^® 568 (Thermo Fisher; #A10042, RRID:AB_2534017), donkey anti-chicken Alexa Fluor^® 488 (Dianova, Germany; #703-545-155, RRID:AB_2340375), donkey anti-guinea pig Alexa Fluor^® 647 (Dianova; #706-605-148, RRID:AB_10895029) were used, each at dilution of 1:500 in PTwH.