K8008

Tissues were fixed in 4% buffered formalin, descaled by EDTA and embedded in paraffin. For immunohistochemical staining^{19 (link)}. Two micrometers of bone marrow tissue sections were incubated with EnVision Flex Target Retrieval Solution, pH low (K8005, DAKO) and stained with primary antibodies directed against SAMHD1 (12586-1-AP, Proteintech, 1:3000) and against CD34 (IR632, DAKO) for 40 min at room temperature. Polymeric secondary antibodies coupled to HRPO peroxidase and DAB were used for visualization (REAL EnVision Peroxidase/DAB +, K5007, DAKO). Tissue samples were analyzed by light microscopy after counterstaining with Meyer’s hematoxylin (K8008, DAKO). Two pathologists, who were blinded to clinical history and therapeutic response, independently scored the SAMHD1 IHCs. They evaluated all tissue sections for nuclear SAMHD1 staining using a four-stage staining score: 0 = negative; 1 = weak intensity of staining; 2 = strong intensity of staining in <25% of blasts; and 3 = strong intensity of staining in more than 25% of blasts. IHC staining scores of 0 and 1 were defined as ‘no or low expression’ and IHC staining scores of 2 and 3 were defined as ‘high SAMHD1 expression’. Membranous CD34 staining for the quantification of the number of AML blasts was evaluated using a two-stage staining score: 0 = negative; 1 = positive.

Rabbit polyclonal antibody specific for PRR [ref. HPA003156; Sigma-Aldrich (Saint Louis, MO, USA) at 1/50 dilution] was used for the immunostaining of formalin-fixed and paraffin-embedded tumour tissues. The antibody’s specificity was tested previously [27 (link)] and the immunostaining process was performed following routine methods in an automatic immunostainer (Dako Autostainer Plus, Dako-Agilent, Santa Clara, California, USA). Briefly, antigen retrieval was carried out in a low-pH buffer (K8005, Dako, Santa Clara, California, USA) for 20 min at 95 °C. The samples were incubated with the primary antibody for 50 min at room temperature. Then, the primary antibody was washed and samples were incubated for 20 min with secondary anti-rabbit antibody (K8021, Dako). EnVision-Flex detection system together with an HRP-enzyme-labelled polymer (SM802, Dako) was employed. A positive reaction was visualized with diaminobenzydine (DAB) solution (DM827, Dako), followed by counterstaining with haematoxylin (K8008, Dako).
Slides were reviewed under light microscopy for staining evaluation. Two observers independently evaluated the slides and, in the event of discrepancies, samples were re-evaluated to arrive at a final conclusion. As previously reported [27 (link)], staining patterns were scored as negative, weak and intense cytoplasmic and membranous staining.