Leica sp8 confocal microscope

To stain bioluminescent L. plantarum in PPs, PPs were frozen in precooled iso-propane, sectioned at 5 mm, and processed for immuno-histochemical staining. As primary antibody, we applied Anti-Firefly Luciferase antibody ab181640 (Abcam) in a 1:250 dilution. As second antibody, we applied FITC-conjugated rabbit anti-goat antibody (1:100, Dako) and as third biotinylated swine anti-rabbit antibody (1:100, Dako). Tissue slides of 2 Å~ were then washed for 10 min with PBS and incubated with DAPI solution (30 nM) for 5 min to visualize nuclei of the epithelial cells. After washing with PBS, samples were mounted with mounting medium. Slices were studied with a SP8 Leica confocal microscope (Leica Microsystems, Son, the Netherlands).

Paraffin sections were rehydrated and washed for 5 min for each step using xylene, 100% ethanol, 95% ethanol, 70% ethanol, H₂O, and PBS. Heat-induced epitope retrieval was performed with sodium citrate buffer (pH 6.0, in PBS). Sections were permeabilized using 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA) in PBS (PBST), then blocked for 1 h at room temperature using 10% normal donkey serum and 0.25% Triton X-100 in PBST. Sections were incubated with primary antibody diluted in blocking buffer overnight at 4 °C. The following primary antibodies were used: MATR3 C-term (rabbit polyclonal, ab84422, 1:400, Abcam, Cambridge, UK), NeuN (mouse monoclonal, MAB377, 1:200, Millipore, Burlington, VT, USA), CTIP2 (rat monoclonal, ab18465, 1:250, Abcam, Cambridge, UK). Sections were washed with PBST 3 times, 5 min each, and incubated with secondary antibodies (488/555 Alexa Fluor donkey anti-rabbit/mouse IgG (H + L), 1:500 from ThermoFisher Scientific, Waltham, MA, USA) and DAPI (D6210, 1:1000, Millipore, Burlington, VT, USA) for 1 h at room temperature. Sections were washed with PBST 3 times, 5 min each, and mounted with ProLong Gold Antifade mountant (ThermoFisher Scientific, Waltham, MA, USA P36930). All slides were imaged using an SP8 Leica confocal microscope (Leica Microsystems, Wetzlar, Germany).

Another part of ileum and colon was fixed in Carnoy’s fixative to preserve the mucus layer. Samples were embedded in paraffin and afterward 4 µm sections of tissue were made with a microtome (Leica Biosystems, Nussloch, Germany). Paraffin was removed from the tissue slides by washing 2× with xylene for 10 min. Next, samples were washed in decreasing amounts of ethanol (99, 70, 60%) and finally in dH₂O. After washing, samples were incubated with 0.5 µg universal bacterial probe EUB338 (5′-GCTGCCTCCCGTAGGAGT-3′) or segmented filamentous bacteria (SFB)-specifc probe SFB1008 (5′-GCGAGCTTCCCTCATTACAAGG-3′) conjugated on the 5′ end with Alexa 488 (Eurogentec, Maastricht, the Netherlands) in hybridization solution (20 mM TRIS-HCl, pH 7.4, 0.9 M NaCl, 0.1% weight/volume SDS) at 50°C overnight in a humid environment. The next day, the samples were washed in FISH washing buffer (20 mM TRIS-HCl, pH 7.4, 0.9 M NaCl) for 20 min. Tissue slides were then washed 2× for 10 min with PBS and incubated with DAPI solution (30 nM) for 5 min to visualize nuclei of the epithelial cells. After washing 2× with PBS, samples were mounted with mounting medium. Data were acquired with a SP8 Leica confocal microscope (Leica Microsystems, Son, the Netherlands), and images were processed afterward with Imaris software (Bitplane, Zurich, Switzerland).

Fifty-micrometer free-floating brain sections were washed twice with 1× PBS prior to incubating with blocking solution (5% normal donkey serum and 0.3% Triton X-100 in 1× PBS) for 1 h at RT. Sections were then incubated in primary antibody diluted in blocking solution for 48 h at 4 °C. The following primary antibodies were used: anti-IBA1 antibody (rabbit polyclonal, Wako-01919741 1 : 1000) and anti-GFAP antibody (rabbit polyclonal, ab7260 1 : 500). Sections were rinsed with 1× PBS and then incubated with secondary antibody (488 Alexa Fluor donkey anti-rabbit IgG (H + L), 1 : 500 from ThermoFisher Scientific) and DAPI (Millipore D6210, 1 : 1000) for 48 h at 4 °C. Sections were rinsed and then mounted with Prolong Gold Antifade mountant (ThermoFisher Scientific, P36930). All slides were imaged using SP8 Leica confocal microscope (Leica Microsystems, Wetzlar, Germany).

TA muscles were teased into thin pieces before being permeabilized with 2% PBST for 30 min, then blocked with blocking solution (4% bovine serum albumin and 1% Triton X-100 in 1× PBS) for 30 min at RT. The tissues were then incubated in primary antibody diluted in blocking solution overnight at 4 °C. The following primary antibodies were used: anti-synaptophysin antibody (rabbit polyclonal, Synaptic Systems 101 002, 1 : 200), anti-synapsin I antibody (rabbit polyclonal, ab64581, 1 : 200), and anti-neurofilament H antibody (chicken polyclonal, ab4680, 1 : 200). After washing, tissues were incubated with secondary antibody (488 Alexa Fluor donkey anti-rabbit or 488 Alexa Fluor goat anti-chicken, 1 : 250 from ThermoFisher Scientific), α-bungarotoxin (Alexa Fluor 555 conjugate, Invitrogen B35451, 1 : 500), and DAPI (Millipore D6210, 1 : 1000) for 1 h at RT. Tissues were then washed and mounted with Prolong Gold Antifade mountant (ThermoFisher Scientific, P36930). Slides were imaged using SP8 Leica confocal microscope (Leica Microsystems, Wetzlar, Germany).