Pannoramic scan slide scanner

Serial sections of 4 mm thickness were taken from representative FFPE tissue blocks, affixed to 3‐aminopropyl triethoxysilane‐coated slides, and air‐dried overnight at 37°C. The sections were then subjected to HE and immunohistochemical staining. Immunohistochemical staining was performed using the following immunoperoxidase polymer methods. After dewaxing and antigen retrieval, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 10 min. After blocking with goat serum, the sections were incubated for 30 min at 37°C with rabbit anti‐CLDN18 (1:500 dilution, clone ab203563; Abcam), rabbit anti‐CDX2 (1:50 dilution, clone A20222; ABclonal Technology Co), rabbit anti‐PAX8 (1:100 dilution, clone bs‐1201R; BIOSS ANTIBODIES), mouse anti‐CEA (1:300 dilution, clone 12‐140‐10; ZSGB‐BIO), mouse anti‐p53 (1:300 dilution, clone DO‐7; ZSGB‐BIO), and mouse anti‐P16 (1:100 dilution; clone MX007, MXB). The slides were visualized with diaminobenzidine (DAB) and counterstained with hematoxylin. Both membranous and nuclear CLDN18 levels were evaluated. The stained slides were scanned with a Pannoramic SCAN slide scanner (3DHISTECH Ltd.) to obtain a whole slide image.

Intact GBM-bearing brains were fixed in 4% paraformaldehyde (PFA) and then embedded in paraffin. Each brain was sliced in the same orientation as the MRI and 5 μm-thick sections were stained with hematoxylin and eosin (H&E). H&E slides were digitally scanned using the Pannoramic SCAN slide scanner (3DHistech) with a 20× objective. Images were viewed and processed using CaseViewer (3DHISTECH, Budapest, Hungary).

Tissue slices were processed according to the standard Deparaffinization and H&E Staining and Decrosslinking protocol (10x Genomics, CG000409). Briefly, slices were deparaffinized and stained with hematoxylin, followed by bluing and eosin staining. Slices were covered with mounting medium (85% glycerol) and coverslipped before imaging. Slides were imaged using the Pannoramic SCAN slide scanner (3D Histech) controlled by the Pannoramic Scanner Software. Straight after imaging, coverslips were removed and samples were directly processed according to the standard Visium Spatial Gene Expression protocol (10x Genomics, CG000407) using the Visium Spatial Gene Expression Slide and Reagent Kit (10x Genomics, PN-1000185). Decross-linking was then performed, and probes were immediately hybridized, ligated, released, and collected for library construction following manufacturer’s recommendations (10x Genomics, CG000409). Tissue-covered spots were quantified, and libraries were pooled according to their concentration and spot occupation on slides. Library pool was sequenced using NovaSeq 6000 (Illumina) using the recommended parameters: read 1, 28 cycles; i7 index, 10 cycles; i5 index, 10 cycles; and read 2, 50 cycles.