2.7 Immunostaining and multiplex immunohistochemistry (mIHC)
Cells in the LOC channels were fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature (RT) after rinsing with PBS. They were then permeabilized with 0.2% Triton-X for 15 minutes and blocked with 5% bovine serum albumin (BSA) for 1 hour at RT. Subsequently, the samples were incubated overnight at 4°C with primary antibodies: CD31 (diluted 1:400, cat no. 281583, Abcam, Cambridge, MA, USA) and E-cadherin (diluted 1:200, cat no. 231303, Abcam). After three washes with PBS, fluorescently labeled secondary antibodies were applied for 1 hour at room temperature: donkey anti-rabbit IgG H&L (Alexa Fluor® 647) (diluted 1/200, cat no. ab150075, Abcam) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (2ug/ml, cat no. ab150117, Abcam). Following additional washes, cells were stained with DAPI for 5 minutes. The membranes containing the cells were then removed from the chip and mounted on cover glass for confocal microscopy imaging.
As for the method of multiplex immunohistochemistry (mIHC), the lung organoid derived epithelial cells on the chip were fixed in 4% PFA for 20 min at RT. The lung organoids were fixed with 4% PFA for 2h at RT after discarding Matrigel. Then, they were mixed with a special embedding gel (SEgel, Suzhou Jiyan Biotech. Co. Ltd., China) for 30 min at 37°C for solidification. These organoids in the SEgel were further fixed with 4% PFA overnight at RT, followed by gradient dehydration with 20%, 30%, and 40% sucrose. After dehydration, they were embedded in optimal cutting temperature compound (OCT, Japan) for frozen sectioning. The chip sample and organoid slides were treated by antigen retrieval through microwave treatment. Endogenous peroxidase activity was quenched, and the samples were then blocked with 5% BSA for 30 minutes. Subsequently, the samples were incubated with primary antibodies overnight at 4°C. After three washes with 1X PBS buffer, the samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 50 minutes at RT. Following a quick wash in 1X PBS buffer, an appropriate fluorophore-conjugated tyramide signal amplification (TSA) was applied for 10 minutes at RT. Microwave treatment was performed to strip the tissue-bound primary/secondary antibody complexes, preparing the samples for labeling with the next marker. This process was repeated for primary antibodies (SFTPC, 1:800, cat no. GB114059, Servicebio; E-cadherin, 1:400, cat no. 3195S, CST), HRP-conjugated secondary antibodies (cat no. WAS1201100, World Advanced Science Co., Ltd., China), and fluorophore-conjugated TSA (TSA570, WAS1003050; TSA520, WAS1002050) until all markers were labeled. Finally, the samples were stained with DAPI in the dark for 10 minutes at RT and mounted with an anti-fade mounting medium. The images were captured using the PANNORAMIC MIDI II imaging system. The lung organoid epithelial cells on the chip were imaged with confocal microscopy (Leica, Germany).