Opal polaris 7 color automation ihc detection kit

IF assays were performed in cells, organoids, and tumor tissues from humans. Cell lines and organoids were fixed with 4% paraformaldehyde for 15 min and then blocked with 5% BSA for 30 min. The primary antibodies were diluted in 1% BSA and incubated at 4 °C overnight. Then, secondary antibodies were added to the samples and incubated at room temperature for 1 hour. The cell nuclei were stained with DAPI. For tumor tissues, samples were soaked in xylene for 15 min twice and then washed with ethanol and anhydrously denatured according to a concentration gradient. Antigen retrieval was conducted with citrate (pH = 6.0), and samples were blocked. The primary antibodies were incubated with the samples at 4 °C overnight. The Opal™ Polaris 7 Color Automation IHC Detection Kit (Akoya) was used according to the protocol.

We performed the multiplex immunofluorescence experiments by using the CD8 anti‐human antibody (Ab) (ab101500), EPCAM anti‐human Ab (Abcam, ab223582), cytokeratin‐8 anti‐human Ab (Abcam, ab53280), cytokeratin‐19 anti‐human Ab (Abcam, ab76539), collagen‐IV (COL4A1) anti‐human Ab (Abcam, ab214417), SDC1 anti‐human Ab (Abcam, ab128936), CD3 anti‐human Ab (Abcam, ab16669), CD56 anti‐human Ab (Abcam, ab75813), CCL22 anti‐human Ab (Abcam, ab23772) and 4′,6‐diamidino‐2‐phenylindole staining solution (Abcam, ab228549). Following the manufacturer's instructions (Akoya, Opal Polaris 7 Color Automation IHC Detection Kit), we scanned the slides with the Akoya Vectra Polaris Automated Quantitative Pathology Imaging System and quantified the results by using Akoya Inform software.

Antibody targeted SMAD3 was used for detecting the following protein: (rabbit, 1:100, Cell Signaling Technologies, #9513, incubation for 30 min at RT).
Furthermore, the Akoya Opal Polaris 7 Color Automation IHC Detection Kit (Akoya, NEL871001KT) was used for the tyramide signal amplification according to the manufacturer’s protocol. All dewaxing and staining steps were performed using the Leica Bond RX slide stainer with the standard settings of the Opal 7‐color (v5.2 plus) IHC protocol provided by the manufacturer. The steps specific for the staining is as follows: Antigen retrieval was performed for 20 min at 100°C with Bond TM Epitope Retrieval 1 solution (Leica, AR9961). Tissue was blocked for 15 min using a blocking buffer (TBS with 1% BSA (VWR, 22013, bovine serum albumin (BSA), fraction V, biotium (50 g))) with 10% normal goat serum (Invitrogen, 10000C). For introduction of the secondary‐HRP, the Envision+/HRP goat anti‐Rabbit (Dako Envision + Single Reagents, HRP, Rabbit, Code K4003) was used for the antibody raised in rabbit (SMAD3). The protein SMAD3 was detected using the OPAL 690 reagent.
After the staining in the Leica Bond RX, slides were washed twice for 2 min in TBS‐Tween‐20 (0.5%) and were then mounted using ProLong Diamond Antifade Mountant (Thermo Fisher, P36961). Numbers of count cells are indicated in Appendix Fig S2 source data file.