Goat anti rabbit

We first divided the mice into an ICH group and a PBS group and observed iba1 antibody cells and MPO staining at 6, 12, and 24 h. Six C57 mice were used in each group. We then used the CX3CR1 + /GFP mice for MPO immunofluorescence with six mice per group. We used 40 μm-thick brain slices for immunohistochemistry. For iba1 staining, we used the ABC method. The antibodies were Rabbit anti iba1 (Solarbio Life Sciences, Beijing, China, 1:600), Goat anti Rabbit (Solarbio, 1:300), and streptavidin–horseradish peroxidase conjugate (STR-HRP). For MPO fluorescence staining, we used Rabbit anti MPO (Solarbio, 1:600) and Goat anti Rabbit (Solarbio, 1:300). Following each antibody staining, the brain slices were washed three times with PBST (PBS + Triton). Images of the immunohistochemically labeled sections and the fluorescently immunolabeled sections were obtained using an Olympus CKX41 fluorescence inverted microscope (Olympus, Tokyo, Japan). The images were captured in the FITC and TRITC channels using Cell-P imaging software (Olympus). When the two images were merged, the cells appeared yellow.

Fresh peripheral blood (5 mL) was collected from CLL patients and healthy controls. Peripheral blood mononuclear cell (PBMC) was isolated using a lymphocyte separation solution (TBD Science). Peripheral blood lymphocytes were lysed in radioimmunoprecipitation assay buffer with protease; phosphatase inhibitors, as well as phenylmethanesulfonylfluoride and the supernatant, were collected after centrifugation. The protein concentration was determined by the bicinchoninic acid method. Sodium dodecyl sulfate (SDS) loading buffer was added to denature the proteins (all the reagents for protein extraction were purchased from Solarbio Company). Proteins were isolated by 12% SDS‐polyacrylamide gel electrophoresis and transferred to the polyvinylidene fluoride (PVDF) membrane (300 mA, 90 min). PVDF membrane was blocked for 1 h by 5% skimmed milk and then incubated with a polyclonal rabbit anti‐Tim‐3 (Proteintech Group) overnight at 4°C. Goat anti‐rabbit (Solarbio) horseradish peroxidase‐conjugated secondary antibody was incubated at room temperature for 1 h. β‐Actin (Cell Signaling Technology) in equal quantities was used as an internal reference. The enhanced chemiluminescence (Biogot Technology) method evaluated the protein expression level.

RIPA lysis buffer (Solarbio, Beijing, China) to extract total protein. The proteins were quantified by the bicinchoninic acid (BCA) assay kit (Solarbio, Beijing, China). Add SDS loading buffer to the extracted total protein, and then boil it in 100° water for five minutes for subsequent experiments. The obtained protein was electrophoresed in 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). PVDF membrane was used for electroporation. The protein electrophoresis was performed at a stable voltage of 100 V, and the electroporation was performed at a stable current of 300 mA for 90 min. After electroporation, soak the PVDF membrane in 5% skimmed milk and place it on a shaker for half an hour. Then add 5 ml of CCND2 (1:1000), IGF2BP3 (1:1000) (Abcam, CA, USA), RB (1:500), and GAPDH (1:5000) (Cell Signaling Technology, MA, USA) rabbit-derived primary antibody to the PVDF membrane and incubate overnight at 4°. After incubating overnight, collect the primary antibody, add Tris-Buffered Sal ine Tween 20 (TBST) and wash three times for 15 min each time. Add goat anti-rabbit (Solarbio, Beijing, China, 1:5000) and incubate for 1 h, then continue to wash with TBST three times. Finally, add electrochemiluminescence (Solarbio, Beijing, China) liquid to expose in the exposure instrument. The antibodies used in the experiments are shown in Additional file 2: Table S2.