Et1606 31

Tumors, hearts, livers, spleens, lungs, kidneys and brains from the mock-BBZ CAR and Pep42-BBZ CAR groups were embedded in paraffin, and then sliced into sections with a thickness of 4 μm. Sections were deparaffinized, rehydrated, and processed for antigen retrieval. H&E staining was performed on heart, liver, spleen, lung, kidney, and brain tissue sections to observe their structure. The tumor, liver and lung tissue sections were blocked with 10% goat serum and incubated with the indicated primary antibodies at 4 ℃ overnight. Cy3-conjuncted goat anti-rabbit secondary antibody (A10520, Invitrogen), FITC-conjuncted goat anti-rabbit secondary antibody (F-2765, Invitrogen), and Cy3-conjuncted goat anti-mouse secondary antibody were used (A10521, Invitrogen). The primary antibodies used were as follows: anti-GRP78 antibody (PA1-014 A, Invitrogen), anti-CD3ζ antibody (ET1607-20, Huabio), anti-NESTIN antibody (sc23927, Santa Cruz), anti-CD4 antibody (ET1606-31, HUABIO), and anti-CD8 antibody (ET1609-52, HUABIO).

The finding of immune infiltration was validated by a dual immunohistochemistry staining. A dual immunohistochemistry staining kit (#DS-0003, ZSGB-BIO, China) was used following the manufacturer’s protocols to assess the association of CD8+ T cell and CAFs in GC tissues. The sections of GC tissue, which had been fixed in formalin and embedded in paraffin, were subjected to deparaffinization in xylene 20 minutes after being heated in an oven at 65°C for 2 hours. Following this, they were rehydrated in 100%, 95%, 85%, and 75% alcohol for 2 minutes each. Antigen retrieval was performed with Citrate solution. All slides were blocked with goat serum buffer at 37°C for 30 min and then incubated with CAFs marker Anti-ACTA2 (1:100, Genxspan, #GXP6460) and CD8 (1:100, Huabio, # ET1606-31) primary antibodies at 4°C overnight. The next day, the slides were incubated with AP-labeled Rabbit and HRP-labeled mouse secondary antibodies at 37°C for 1 hours. Then, the related products were detected with DAB and RED respectively. The nuclei were stained for 1 to 2 minutes using hematoxylin. Finally, the sections were dehydrated, transparent and sealed with gum. The slides were viewed with a microscope and images captured.