Anti ly6g

Formalin-fixed and paraffin-embedded tissues sectioned to 4 μm were used for histological evaluation of liver tumors in a mouse model. Hematoxylin and eosin (H&E) staining was performed for each sample. For IHC, tissue slides were deparaffinized with xylene and rehydrated through a graded series of ethanol solutions (100%, 95%, and 70%). Subsequently, the slides were subjected to antigen retrieval by microwaving in a citric acid solution for 15 min. The primary antibodies anti-hepatocyte (Abcam, Cat#ab75677), anti-Epcam (Abcam, Cat#ab213500), anti-CD3 (Abcam, Cat#ab16669), anti-Ly6G (Servicebio, Cat#GB11229), anti-CD8 (Abcam, Cat#ab217344), anti-F4/80 (CST, Cat#70076), anti-Asma (Servicebio, Cat#BM0002), and anti-CD31 (Servicebio, Cat#GB113151) were used. Subsequently, the slides were incubated with secondary antibodies (1:1, 100 μL for each slide; HRP-anti-rabbit IgG, ZSGB, Cat#PV-6001, or HRP-anti-mouse IgG, ZSGB, Cat#PV-6002) for 10 min at room temperature. Multispectral images were scanned with a ZEISS AXIOSCAN 7 instrument. Cells of interest were quantified in Halo v3.4 (Indica Labs) or QuPath v0.2.0. Each section was evaluated by 2 or 3 experienced pathologists.

The paraformaldehyde-fixed livers were dehydrated, embedded in paraffin, and cut into 5- to 7-μm-thick sections before being subjected to H&E staining as described previously.¹² (link) The severity level of liver damage was assessed for inflammatory infiltration, cell swelling, and tissue architecture disruption in a blinded fashion and scored on a 4-point scale (0, none; 1, slight; 2, moderate; 3, severe).
For immunofluorescence assays, the liver sections were incubated with anti-CD31 (1:3,000, Proteintech, IL, USA), anti-Glut1 (1:100, Proteintech), anti-Sele (1:2,000, Proteintech), anti-Ly6G (1:3,000, Servicebio, Wuhan, China), anti-Lta4h (1:1,000, Proteintech), anti-Sort1 (1:400, Proteintech), anti-ATF4 (1:1,000, ABclonal, Wuhan, China), anti-Fosl1 (1:1,000, ABclonal), anti–NF–κB1(1:1,000, ABclonal), and anti-F4/80 (1:500, Servicebio) primary antibodies, followed by washing and incubation with the fluorophore-labelled secondary antibody, and visualisation using a confocal microscope (Nikon, Tokyo, Japan).

The specimens were fixed in 4% neutral buffered formalin and then embedded in paraffin. 4 μm-thick Liver sections were stained with hematoxylin and eosin. The severity of liver IR was graded using Suzuki’s criteria on a scale from 0 to 4, or with antibodies using standard immunohistochemistry protocols. For Immunofluorescence, the fixed tissue sections were placed in a repair kit (Servicebio, China) filled with EDTA antigen retrieval buffer (PH = 9.0), and then boiled in a microwave for repair. After natural cooling, the slides were washed three times with PBS. BSA (3%) in PBS solution was added for 30 min to block nonspecific binding, and then the corresponding primary antibodies were added: anti-SGK1(23394–1-AP, Proteintech), anti-p-STAT3 (Tyr705) (bs-1658R, Bioss), anti-HNF-4α (ab201460, Abcam), anti-IL-6 (Servicebio, China), anti-MPO (ab208670, Abcam), anti-CitH3 (ab281584, Abcam), anti-CD36 (Servicebio, China), anti-Ly6G (Servicebio, China), and anti-SAA (Servicebio, China). After overnight incubation, the secondary antibody against the corresponding species was added for 50 min in the dark. DAPI was used for the nuclear counterstaining. Slides were observed under a confocal fluorescence microscope (NIKON ECLIPSE C1) [35 (link)].