Carnoy solution

The tissue was collected in the same manner as for RNA, placed in cassettes and fixed in 10% formalin for H&E staining or Carnoy solution (ThermoFisher) for fluorescent in situ hybridization (FISH) staining overnight at room temperature. Cassettes were transferred to 70% ethanol for formalin or 100% ethanol after Carnoy fixation to wash out the fixative. The tissue was embedded in paraffin, and slides were cut at 5 μm thickness. The H&E staining was performed by the Human Tissue Resource Center at the University of Chicago. For FISH staining, the paraffin was first removed by running the slides through four 3-min incubations in xylene and four 3-min incubations in 100% ethanol. The slides were then moved to a polypropylene slide container and filled with hybridization solution containing the diluted 16S probe (0.9M NaCl, 20mM Tris-HCL pH 7.5, 0.1% SDS with 0.2ng of probe specific for SFB 16S or universal 16S) ^{68 (link)}. The 16S probes used are included in Key resources table. The slides were incubated overnight at 50 °C in the dark. The slides were washed three times with the hybridization buffer, briefly rinsed in H₂0, and then mounted with Prolong diamond antifade with DAPI (ThermoFisher). The slides were scanned with the CRi Pannoramic SCAN 40x Whole Slide Scanner at the University of Chicago Integrated Light Microscopy core.

The tissue was collected in the same manner as for RNA, placed in cassettes and fixed in 10% formalin for H&E staining or Carnoy solution (ThermoFisher) for fluorescent in situ hybridization (FISH) staining overnight at room temperature. Cassettes were transferred to 70% ethanol for formalin or 100% ethanol after Carnoy fixation to wash out the fixative. The tissue was embedded in paraffin, and slides were cut at 5 μm thickness. The H&E staining was performed by the Human Tissue Resource Center at the University of Chicago. For FISH staining, the paraffin was first removed by running the slides through four 3-min incubations in xylene and four 3-min incubations in 100% ethanol. The slides were then moved to a polypropylene slide container and filled with hybridization solution containing the diluted 16S probe (0.9M NaCl, 20mM Tris-HCL pH 7.5, 0.1% SDS with 0.2ng of probe specific for SFB 16S or universal 16S) ^{68 (link)}. The 16S probes used are included in Key resources table. The slides were incubated overnight at 50 °C in the dark. The slides were washed three times with the hybridization buffer, briefly rinsed in H₂0, and then mounted with Prolong diamond antifade with DAPI (ThermoFisher). The slides were scanned with the CRi Pannoramic SCAN 40x Whole Slide Scanner at the University of Chicago Integrated Light Microscopy core.