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).