Luxol fast blue (lfb)

The following reagents were used during the course of the experiments and were purchased as following: red blood cell (RBC) lysis buffer was purchased from Millipore Sigma (Burlington, MA); Percoll from GE Healthcare Life Sciences (Pittsburgh, PA); Myelin oligodendrocyte glycoprotein peptide subunit 35–55 (MOG35-55) from PolyPeptide Laboratories (San Diego, CA); Pertussis Toxin (PTX) from List Biological Laboratories (Campbell, CA); Heat killed Mycobacterium tuberculosis (H37Ra) from Difco (Detroit, MI); β-mercaptoethanol, Freund’s Advjuvant, propionic acid, n-butyric acid, isovaleric acid, 2-ethylbutyric acid, sulfuric acid and Tween-80 from Sigma-Aldrich (St. Louis, MO); absolute ethanol, acetic acid, lithium carbonate and tryptamine were purchased from Fisher Scientific (Hamptom, NH); RPMI 1640, fetal bovine serum (FBS), L- glutamine, phosphate buffered saline (PBS), luxol fast blue (LFB), cresyl violet, eosin, hematoxylin, and HEPES were purchased from VWR (West Chester, PA; flourophore conjugated antibodies and ELISA kits from Biolegend (San Diego, CA); Illumina MiSeq reagents from Illumina Inc. (San Diego, CA); QIAamp Stool Mini Kits from Qiagen (Germantown, MD); 10% formalin from Azer Scientific (Morgantown, PA).

Paraffin sections were stained with Luxol Fast Blue (VWR, Radnor, PA, USA) for oligodendrocyte myelin followed by with Cresyl Violet (Sigma, St. Louis, MO, USA) for neuronal Nissl bodies’ assessment. Sections were rehydrated in xylene for 20 min and decreasing concentrations of ethyl alcohol for 10 min each. Sections were then incubated with a 0.1% Luxol Fast Blue solution in 70% ethyl alcohol overnight at 56°C. After rinsing with 70% ethyl alcohol followed by distilled water to remove excess stain, the tissue was differentiated in a 0.5% lithium carbonate solution. Slides were then rinsed in distilled water followed by 2% acetic acid solution for 5 min. Next, tissue was counterstained with a 1% Cresyl Violet solution for 10 min. After rinsing in distilled water, sections were differentiated in 37.5% acetic acid and rinsed in distilled water. Slides were mounted with Fluoromount-G (Southern Biotech, Birmingham, AL, USA) and visualized using a DFC 490 camera (Leica, Wetzlar, Germany) adapted to an Axioskop bright field microscope (Zeiss, Oberkochen, Germany). Width of cerebellar layers, number of neurons per field, and the area of their soma were analyzed using ImageJ software (NIH, Bethesda, MD, USA). Intensity of staining of the external germinal layer at LPS1 and of the white matter layer at LPS9 was analyzed using the same software and was expressed per square micrometers.