Alexa fluor 488

Spinal cords were dissected, rapidly frozen on dry ice, and embedded in Tissue-Tek (Sakura Finetek). Transverse slices with a thickness of 8–9 μm were cut with a cryostat (Jung Frigocut 2800E, Leica). Four to six slices were transferred to glass slides (25 × 75 × 1.0 mm; SuperFrostR Plus, Menzel Gläser/Thermo Scientific) and stored for further analysis at −80°C. For immunostaining, tissue slides were fixed for 30 s in ice-cold 2% PFA and immersed once in 50 mm NH₄Cl following quenching for 30 min in 0.1 mm glycine in PBS. Tissue slices were blocked by 10% normal horse serum in PBS for 1 h at room temperature. The slides were incubated with antibodies GlyR α1 (1:250; mAb2b; catalog #146 111, Synaptic Systems), VGAT (1:300; catalog #131 003, Synaptic Systems), gephyrin (1:200; catalog #147 003, Synaptic Systems), and synapsin (1:300; catalog #574778, Calbiochem) in 10% normal horse serum in PBS overnight at 4°C. After three washing steps with PBS for 10 min each, tissue was incubated with secondary antibodies coupled to Cy3 and Alexa Fluor 488 (1:1000, Dianova) diluted in 10% normal horse serum in PBS for 45 min at 22°C. For staining of the cell nuclei, slides were incubated in Molecular Probes DAPI solution (Thermo Fisher Scientific) diluted 1:1500 in PBS for 10 min at room temperature in a dark chamber. Finally, the slides were mounted with aqua polymount (Polysciences).

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).

14 days after immunization, eyes were fixed in 4% paraformaldehyde (PFA) for 1 h and then prepared as flatmounts (n = 6/group). The following steps were performed at 20°C on a thermo shaker (70 rpm). First, the flatmounts were blocked with 10% donkey serum and 0.5% Triton-X in PBS for 90 min. Then, they were incubated with the RGC marker Brn-3a (Nadal-Nicolas et al., 2009 (link)) (1:300; Santa Cruz, CA, USA) overnight, followed by a 2 h incubation of donkey anti-goat Alexa Fluor 488 (1:1000; Dianova, Hamburg, Germany). From each of the four flatmount arms, three photos were captured (central, middle, and peripheral) with an Axiocam HRc CCD camera on an Axio Imager M1 fluorescence microscope (Zeiss, Jena, Germany). Cells were counted using ImageJ software (NIH, USA). Group comparison was performed after transferring the data to Statistica software (V10.0; Statsoft, Tulsa, OK, USA).

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.

At 15 weeks, eyes were fixed in 4% paraformaldehyde for 1 hour and then prepared as flatmounts (n = 12 eyes/group).18 Briefly, flatmounts were blocked with 10% donkey serum in 0.5% Triton‐X in PBS for 90 minutes. Afterwards, they were incubated with the RGC marker Brn‐3a (1:300, Santa Cruz) overnight. The corresponding secondary antibody, donkey anti‐goat Alexa Fluor 488 (1:1000, Dianova), was added the next day for 2 hours. From each of the four flatmount arms, three photos were captured (central, middle, peripher) with an Axio Imager M2 fluorescence microscope (Zeiss). Brn‐3a⁺ cells were counted using ImageJ software (NIH).