Saturated lithium carbonate

Mouse brains at P5, P10 and P14 were dissected and fixed in Bouin’s fixative for 18–24 hours at room temperature (RT). Brains were rinsed in tap water followed by washes in 70% ethanol with saturated lithium carbonate (Sigma-Aldrich, St. Louis, MO). Tissue was then dehydrated in a graded series of ethanol washes, embedded in paraffin and sectioned into 8–10 μm sections using a Leica Microsystems RM2125 microtome (Leica, Wetzlar, Germany). All sections shown are coronal unless stated otherwise. Paraffin was removed though xylene washes and sections rehydrated in a series of ethanol washes. Brain sections were stained using Harris hematoxylin and alcoholic eosin Y solution (Sigma-Aldrich, St. Louis, MO). Slides were subsequently dehydrated, washed in xylene and mounted using Cytoseal XYL (Fisher Scientific, Pittsburgh, PA). Imaging was performed using a Leica MZFLIII dissecting microscope equipped with a Leica DFC320 color camera (Leica, Wetzlar, Germany).

Brown and Brenn (B&B) staining was done as previously described [10 (link)]. Slides were deparaffinized (xylene (Gadot, CAS 1330-20-7) 10 min twice) and rehydrated with descending concentrations of ethanol (100%—5 min, 90 and 70%—2 min each) to tap water (for 10 min). Thereafter, they were stained with a sequence of the following solutions; Harris haematoxylin (Sigma HHS32)—5 min, acid alcohol (1% HCl in 70% ethanol)—5 s, saturated lithium carbonate (Sigma 62470) in DDW—5 s, Hucker’s solution (0.2% crystal violet (sigma 61135)), 2% ethanol, 0.8% ammonium oxalate ((sigma 221716) in DDW)—2 min, iodine solution (0.33% iodine (BDH CAS 7553-56-2)), 0.66% potassium iodide ((J.T. Baker CAS 7681-11-0) in DDW)—15 s, ether acetone (50% Ether (Daejung CAS 60-29-7)), and 50% acetone (Gadot, CAS 67-64-1)—10 s, basic fuchsin (0.007% basic fuchsin (Sigma 857343, 7.31% ethanol in DDW))—3 min, acetone—10 s, picric acetone (0.1% picric acid in acetone EMS 26105-06)—20 s, acetone–xylene I (33.3% acetone, and 66.6% xylene)—5 s, acetone–xylene II (25% acetone, 75% xylene)—5 s, and xylene—1 min. The slides were mounted with entelane (mercury 1.07961) and covered with a coverslip. For analysis, each canal was divided into three parts, and evaluation was done by manually determining the most apical localization of bacteria in each canal. Statistics were performed using Fisher’s exact test.