Prechilled hybridization buffer

FISH was performed with a Fluorescent In Situ Hybridization Kit (RiboBio), following the manufacturer’s protocols. Whole mouse brains were dissected and directly incubated overnight in fixing solution (phosphate-buffered saline (PBS) containing 1.6% paraformaldehyde and 20% sucrose), embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek, Torrance, CA, USA), and snap frozen in 2-methylbutylene on dry-ice. The 40-µm sections were washed with ice-cold PBS (twice for 10 minutes), and blocking buffer [tris-buffered saline (TBS) containing 10% bovine serum albumin (BSA)] was added for 30 minutes. Sections were incubated with 0.45 pmol/µL eubacterial oligonucleotide probe in prechilled hybridization buffer (Sigma-Aldrich, St Louis, MO, USA) overnight at 4 °C. Sections were counterstained with 30 nM 4’,6-diamidino-2-phenylindole (DAPI) in PBS for 10 minutes, washed for 10 minutes in ice-cold PBS, and mounted with Fluoromount-G. Immunofluorescent imaging was performed on the same day.

Whole ceca were dissected and directly incubated overnight in fixing solution (PBS containing 1.6% paraformaldehyde and 20% sucrose), embedded in O.C.T. (Tissue-Tek), and snap frozen in 2-methylbutylene on dry-ice. 10-µm sections were washed with ice-cold PBS (twice for 10 min), and blocking buffer (TBS containing 10% BSA) was added for 30 min. Sections were incubated with 0.45 pmol/µl eubacterial oligonucleotide probe ([AminoC6 + Alexa Fluor 594] 5′-GCTGCCTCCCGTAGGAGT-3′; Operon) in prechilled hybridization buffer (Sigma-Aldrich) overnight at 4°C (Bates et al., 2006 (link)). Sections were counterstained with 30 nM DAPI (Invitrogen) in PBS for 10 min, washed for 10 min in ice-cold PBS, and mounted with Fluoromount-G. Immunofluorescent imaging was performed on the same day.