Sap 9100

After the section of DLBCL paraffin tissues, the samples were kept in an oven at 55 °C for 60 min. Dried tissue sections were dewaxed and rehydrated. The dewaxing process was as follows: dimethylbenzene for 10 min, dimethylbenzene for 10 min, absolute ethyl alcohol for 10 min, absolute ethyl alcohol for 10 min, 95% ethyl alcohol for 10 min, 95% ethyl alcohol for 10 min, 85% ethyl alcohol for 10 min, 70% ethyl alcohol for 10 min, and tap water. Antigens were recovered after heating with citrate buffer and 3% H2O2 at high pressure for 10 min, and then sealed with 5% normal goat serum (NGS, SAP-9100, ZSGB-BIO) for 1 min at room temperature, which was then incubated with NGS-diluted PIM kinase family antibodies (PIM1: ab54503, PIM2: ab107102, PIM3: ab198842, USA, Abcam) (dilution, 1:300; Proteintech Group Inc.) overnight at 4 °C 1 h later. NGS was taken as a negative control. The samples were incubated with biotin-labeled goat anti-mouse/rabbit immunoglobulin G (SAP-9100, ZSGB-BIO) for 15 min on the following day, which were, then, incubated and colored with diaminobidine solution, counterstained with hematoxylin for 2 min, and sealed with neutral glue. The expression levels of proteins of PIM kinase family were assessed by the product of the percentage of positive cells and staining intensity.

The bone tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. After incubating in 3% H₂O₂ at room temperature for 10 min followed by antigen recovery, the sections were blocked with 10% goat serum. The sections were then incubated with primary rabbit monoclonal anti-ATF3 (1 : 200; ab254268, Abcam, Cambridge, UK), anti-GPX4 (1 : 200; ab125066, Abcam), or anti-SLC7A11 (1 : 200; ab175186, Abcam) at 4°C overnight. The next day, the sections were incubated with secondary antibodies (SAP-9100, ZsBio, Beijing, China) for 2 h followed by an incubation with 0.1% DAPI for 5 min. After washing with PBS, images were acquired using a microscope (Leica Microsystems), and ImageJ software was used for semiquantitative analysis.

The slices (paraffin sections) of normal brain tissues and tumour tissues were used for immunohistochemistry analysis. Briefly, the slices were dewaxed, gradient dehydrated with alcohol, washed with standard method, repaired with water bath in antigen repairing solution and cooled with tap water. Then, the slices were blocked by normal goat serum solution (SAP-9100, ZSGB-BIO, Beijing, China). Then, the slices were dealt with the anti-IL31RA (K008474P, Solarbio, Beijing, China) and goat anti-rabbit IgG, and the slices were washed with standard methods, respectively. After that, the slices were dripped with Streptomyces ovalbumin protein labelled with horseradish peroxidase, and the slices were color developed with DAB (ZLI-9017, ZSGB-BIO, Beijing, China). Hematoxylin (ZLI-9610, ZSGB-BIO, Beijing, China) was used for counterstaining. Finally, the pathologist observed and interpreted the stained tissues under a light microscope.