Vectashield vibrance mounting media

Immunofluorescence staining was performed as previously described with modifications^{12 (link)}. Slides containing brain sections were first post-fixed in 10% neutral buffered formalin (Fisher Scientific, SF98-4) for 5–10 min, then washed three times with PBS. Antigen retrieval was performed using the Antigen Unmasking Solution (Vector Laboratories, H-3300) at 95 °C for 20–60 min in a water bath with gentle shaking (60 rpm). Slides were cooled to room temperature and permeabilized with 0.3% Triton X-100 in PBS, then blocked with 5% normal donkey serum in 0.3% Triton X-100 in PBS. Slides were incubated at 4 °C overnight with the primary antibodies in blocking buffer. The slides were washed three times with 0.3% Triton X-100 in PBS, then incubated with secondary antibodies in blocking buffer at room temperature for two hours. The slides were then washed twice with 0.3% Triton X-100 in PBS, and then once with PBS. Autofluorescence quenching was carried out to reduce background autofluorescence using the Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, SP-8400) and all slides within one experiment were treated similarly. Slides were counterstained with DAPI and then mounted using VECTASHIELD Vibrance mounting media (Vector Laboratories, H-1700).

Resuspended cells were mounted onto glass slides with Vectashield Vibrance mounting media (Vector Labs, Burlingame, CA) and analyzed using a Leica DM500 fluorescence microscope (Leica, Buffalo Grove, IL) or a Zeiss LSM 880 (Carl Zeiss, Jena, Germany) inverted confocal microscope, all the images were analyzed using Fiji Image J software (45 (link)). Additionally, the supernatant from each microbial isolation from cecal contents was imaged for confirmation of the absence of bacteria with Hoechst 33342 (1 μg/ml) and imaged using a fluorescence microscope. Samples were imaged by using two color filter cubes: UV (359 nm/461 nm) for Hoechst to localize cells and Cy5 (650 nm/670 nm) for AF647 to detect the presence of alkyne-containing metabolites.

After sequence verification, ACR and KCR construct variants were cloned into the multiple cloning sites of a pcDNA3.4 vector (Genscript) by XhoI and EcoRV restriction enzyme digest. Then, 250 ng of the respective DNA constructs were transfected into N2a cells⁵⁴ using Lipofectamine 3000 (Invitrogen). The cells were left to incubate in serum-free media for 48 h. The N2a cultures were then washed three times with PBS and fixed for 20 min at room temperature with 4% paraformaldehyde diluted in PBS-Triton X-100 (0.25%, 85111 Thermo Fisher Scientific). After fixation, the cells were blocked in 5% BSA (A-420-500, Gold Biotechnology) diluted in PBS-Triton X-100 (0.25%) for 1 h at room temperature and stained for GFP (Abcam ab13970, RRID: AB_300798) at 1:2000 v/v dilution for 1.5 h at 37°C. Afterward, the cultures were rinsed three times with PBS and incubated with an Alexa 488 goat anti-chicken (A-11039 Thermo Fisher Scientific, RRID: AB_2534096) at 1:500 v/v dilution for 1 h at 37^oC. Finally, the cells were washed three times with PBS and mounted onto microscope slides in Vectashield Vibrance mounting media (H-1700 Vector Laboratories, Burlingame, CA). Imaging was performed on a Zeiss LSM700 upright microscope using a 100× objective. Maximum intensity projections were obtained after image analysis with ImageJ.