Donkey anti chicken alexa fluor 647

Immunohistochemistry experiments on coronal brain sections (50–100 μm, vibratome, Microm; 14 μm, cryostat, Leica) were carried out as described previously (Cloarec et al., 2016 (link)) with the following primary (anti-Iba1: 1/500, Wako; anti-Cd68, Ed1 clone: 1/200, Millipore) and secondary (Alexa Fluor 568 or 647-conjugated goat anti-rabbit or anti-mouse IgGs; Life Technologies) antibodies. Hoescht 33258 (1:2000, Sigma) was used for nuclei staining.
For tissue clearing experiments (see next subsection), whole infected brains were first immunostained as follows. Tissue samples were dehydrated in methanol/1X PBS series: 20, 40, 60, 80, 100 × 2 for 1h each at room temperature (RT) and then incubated overnight at RT on a platform shaker in a solution of PBSG-T [PBS 1X containing 0.2% gelatin (Sigma-Aldrich), 0.5% Triton X-100 (Sigma-Aldrich) and 0.02% Sodium-Azide (Sigma-Aldrich)]. Next, samples were transferred to PBSG-T containing anti-GFP antibodies (AVES, 1:2,000) and placed at 37°C, with rotation at 100 rpm, for 10 days. This was followed by six washes of 1 h in PBSG-T at RT. Samples were then incubated in secondary antibodies (Donkey anti-chicken Alexa-Fluor 647, Jackson ImmunoResearch, 1:500) diluted in PBSG-T for 2 days at 37°C. After six washes of 1 h in PBSG-T at RT, samples were stored at 4°C in PBS until clearing.

Cells were fixed with paraformaldehyde (4% in PBS) for 20 min, followed by permeabilization (0.2% Triton X-100 in PBS for 10 min) and blocking (3% BSA in 0.2% Triton X-100 in PBS for 10 min). Cells were incubated in primary antibody overnight at 4 °C (Mouse anti-Polyglutamine-Expansion Diseases Marker Antibody (Merck Millipore, MAB1574, 1:50), Rabbit anti-Cleaved Caspase-3 (Cell Signaling, 9661, 1:400), Rabbit anti-DARPP32 (Abcam, ab40801, 1:100), Chicken anti-MAP2 (Abcam, ab5392, 1:1,000). After washing with PBS, cells were incubated in secondary antibody (Alexa Fluor 488 goat anti-mouse (ThermoFisher Scientific, A-11029, 1:1,000), Alexa Fluor 647 donkey anti-chicken (Jackson ImmunoResearch, 703-605-155, 1:1,000), Alexa Fluor 568 goat anti-Rabbit (ThermoFisher Scientific A-21067, 1:1,000)) and co-stained with 2 μg ml⁻¹ Hoechst 33342 (Life technologies). Finally, coverslips were covered with Mowiol (Sigma).