Microm

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.

Immunohistochemistry experiments were carried out as described previously [11 (link)] on coronal brain slices (50–100 μm, vibratome, Microm; 14 μm, cryostat, Leica) with the appropriate primary (Table A in S1 File) and secondary (Alexa Fluor_568 or 647-conjugated goat anti-rabbit, anti-mouse, anti-rat, anti-guinea pig IgGs or donkey anti-goat IgGs; Life Technologies) antibodies. Hoescht 33258 (1:2000, Sigma) was used for nuclei staining.

The tissue was cut with a microtome (Microm, Leica, Nussloch, Germany), deparaffinized and immunohistochemistry was performed with rabbit anti-Ki67 antibodies (IHC-00375, Bethyl Laboratories Inc., Montgomery, TX, USA) to stain proliferating cells. Biotin labeled anti-rabbit antibodies (1:200, Dianova, Hamburg, Germany) and AP-streptavidin (1:200, Vector, Burlingame, CA, USA) were used to visualize Ki67-positive cells in colons. Macrophages were stained with rat anti-F4/80 (Acris # 4007, Rockville, MD, USA), 1:500 and secondary biotinylated anti-rat/streptavidin-AP (Vector, Burlingame, CA, USA), 1:200. T cells were stained with rat anti-CD3 (#DIA-303, Hamburg, Germany), 1:50 and secondary biotinylated anti-rat (Vector), 1:200 and streptavidin- AP (Vector, Burlingame, CA, USA), 1:200. Images were taken with a Keyence BZ-9000 microscope (Neu-Isenburg, Germany).