All samples were reviewed by a genitourinary pathologist (Y.B.C.) to confirm diagnosis. Immunohistochemistry for FH and 2-succino-cysteine (2SC) [FH loss and/or 2SC gain] was performed in 5-mm FFPE tissue sections where tissue was available. Immunohistochemistry for FH was performed using a mouse monoclonal antibody (clone J-13, Santa Cruz Biotechnology) as previously described.(21 (link)) An absence of FH staining in the neoplastic cells, in the presence of positive internal control (cytoplasmic, granular staining in non-neoplastic cells), was interpreted as lost or FH-deficient status. Immunohistochemical staining for S-(2-succino)-cysteine (2SC) was performed using a polyclonal antibody described previously.(10 (link), 22 (link)) Briefly, 4-μm-thick sections from representative formalin-fixed, paraffin-embedded tissue blocks were processed using the Ventana Discovery XT system with antigen retrieval (CC1 solution, 60 min), primary antibody (1:2000), and OptiView DAB IHC detection steps (Ventana). The presence of diffuse, nuclear and cytoplasmic staining was interpreted as positive.
Optiview dab ihc detection steps
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The OptiView DAB IHC detection steps is a laboratory equipment product. It is used for the detection of specific biological targets in tissue samples through immunohistochemistry (IHC) techniques. The product provides a series of steps to enable the visualization of target proteins using a diaminobenzidine (DAB) chromogen.
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Immunohistochemical Analysis of FH and 2SC
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Immunohistochemical Analysis of FH and 2SC
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Immunohistochemical staining for FH was performed using a mouse monoclonal antibody (clone J‐13, Santa Cruz Biotechnology) as previously described (Smith et al., 2016 ). An absence of FH staining in the neoplastic cells, in the presence of positive internal control (cytoplasmic, granular staining in non‐neoplastic cells), was interpreted as lost or FH‐deficient status. Immunohistochemical staining for S‐(2‐succino)‐cysteine (2SC) was performed using a polyclonal antibody as described previously (Bardella et al., 2011 ; Chen et al., 2014 ). Briefly, 4‐μm thick sections from representative formalin‐fixed, paraffin‐embedded tissue blocks were processed using the Ventana Discovery XT system with antigen retrieval (CC1 solution, 60 min), primary antibody (1:2,000), and OptiView DAB IHC detection steps (Ventana). The presence of diffuse, nuclear, and cytoplasmic staining was interpreted as positive.
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