Plastic chamber slides

HaCaT cells located on plastic chamber slides (Thermo Fisher Scientific, Inc., MA, USA) were pretreated with LOC for 1 h followed by treatment with the inducer TNFα/IFNγ (10 ng/ml) for 2 h. After washing with cold PBS, fixation was performed for 10 min using 4% paraformaldehyde in PBS. Next, the cells were treated with 0.1% Triton X-100 (Bio-Rad Laboratories, Inc., WA, USA) in PBS for 10 min and then blocked with 2% bovine serum albumin (BSA) in PBS for 1 h. The primary antibody (NF-κB p65, Cat. No. #8242, Cell Signaling Technology, Inc., MA, USA; 1 : 800 dilution) was diluted in 1% BSA in PBS and incubated overnight at 4°C. Then, Alexa Fluor 488-conjugated secondary antibody (Cat. No. A-11034, Thermo Fisher Scientific, Inc., MA, USA; 1 : 400 dilution) was incubated for 2 h. The nuclei were stained with DAPI (Hoechst 33342, Thermo Fisher Scientific, Inc., MA, USA) diluted in PBS for 5 min. The slide was mounted (Vectashield® medium, Vector Labs, Inc., Burlingame, CA, USA) and visualized with an LSM 510 Meta system (Carl Zeiss AG, Oberkochen, Germany).

Macrophages were cultured on plastic chamber slides (Thermo Fisher Scientific) and pre-fixed in 2.5% glutaraldehyde under moderate conditions at room temperature. The samples were washed four times with PBS for 3 min before incubation with 2% osmium tetroxide for 30 min at 4 °C. After being washed with distilled water for 5 min, the tissues were dehydrated and 100% EtOH for 30 min, finally, the samples were handstand-embedded for 100% resin. The resins were cured at 60 °C for 3 days. The samples were sliced at 70-nm using an ultramicrotome (Ultracut-UCT, Leica, Wetzlar, Germany). After staining of ultrathin sections with uranyl acetate and lead citrate, TEM observation was performed using H-7600 (Hitachi, Tokyo, Japan) [17 (link)]. The aspect ratio of each mitochondrion was measured with Image-J software by manually tracing outlines of mitochondria on TEM micrographs and calculated as the result of dividing the value of the major axis by the value of the minor axis. Mitochondria from at least 3 sections of 5 cells from each condition were analyzed.

2D colonoids monolayers were grown on plastic chamber slides (Thermo Fisher Scientific, 177445) and fixed with either 4% paraformaldehyde (PFA) or cold 100% ethanol. PFA fixed monolayers were permeabilized with 0.5% Triton X-100 for 10 minutes. Monolayers were blocked with 3% goat or donkey serum in DPBS with 0.05% Tween-20 blocking buffer for 30 minutes. Primary antibodies were diluted in blocking buffer, and cells were incubated overnight at 4°C. Cells were washed with PBS with 0.05% Tween 20, and fluorescently labeled secondary antibodies were diluted in blocking buffer followed by incubation for 1 hour at room temperature. Cells were washed and mounted in Prolong Gold antifade agent (Thermo Fisher Scientific, P36930). Frozen ileal or colonic sections (6–8 μm) from Jam-a^fl/fl and Jam-a^ERΔIEC mice were fixed with 4% PFA, and immunolabeling was performed as described above. Fluorescence imaging was performed with a Nikon A1 confocal microscope (Nikon) in the Microscopy & Image Analysis Laboratory Core at University of Michigan. A detailed list of antibodies used can be found in Supplemental Table 1.