Immunohistochemical Staining of SOX2

Hematoxylin and eosin staining was performed as previously described using a Sakura Tissue-Tek Prima Autostainer (Torrance, CA) [2 (link)]. For IHC staining, formalin-fixed, paraffin-embedded slides were deparaffinized in xylene and hydrated using graded ethanol washes. Tissues were treated with antigen retrieval buffer (S1699 from DAKO; Glostrup, Denmark) in a steamer for 20 min. Anti-SOX2 (D6D9 rabbit monoclonal, Cell Signaling Technology; Danvers, MA) was applied for 1 h at room temperature in a humidity chamber. Following TBS wash, antigen–antibody binding was detected with Envision+system (K4001, DAKO; Carpinteria, CA) and DAB+Chromogen (K3468, DAKO). Tissue sections were briefly immersed in hematoxylin for counterstaining and were cover-slipped. Staining quantitation was conducted by a genitourinary pathologist. Controls for specificity of anti-SOX2 staining using the D6D9 antibody are shown in Supplemental Figure 1.

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de Wet L., Williams A., Gillard M., Kregel S., Lamperis S., Gutgesell L.C., Vellky J.E., Brown R., Conger K., Paner G.P., Wang H., Platz E.E., De Marzo A.M., Mu P., Coloff J.L., Szmulewitz R.Z, & Vander Griend D.J. (2022). SOX2 Mediates Metabolic Reprogramming of Prostate Cancer Cells. Oncogene, 41(8), 1190-1202.

Publication 2021

S1699 Anti-SOX2 (D6D9 rabbit monoclonal, Envision+system DAB+Chromogen

Antibody Antigen Buffer Chromogen Eosin Ethanol Formalin Genitourinary Hematoxylin Humidity Paraffin Pathologist Rabbit Sox2 Tissue Xylene

Corresponding Organization :

Other organizations : University of Chicago, University of Illinois at Chicago, Johns Hopkins University, Sidney Kimmel Comprehensive Cancer Center, The University of Texas Southwestern Medical Center

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Variable analysis

independent variables

Hematoxylin and eosin staining
IHC staining with anti-SOX2 (D6D9 rabbit monoclonal, Cell Signaling Technology)

dependent variables

Staining quantitation conducted by a genitourinary pathologist

control variables

Formalin-fixed, paraffin-embedded slides
Deparaffinization in xylene and hydration using graded ethanol washes
Antigen retrieval using antigen retrieval buffer (S1699 from DAKO) in a steamer for 20 min
Incubation with anti-SOX2 antibody for 1 h at room temperature in a humidity chamber
Detection of antigen-antibody binding with Envision+system (K4001, DAKO) and DAB+Chromogen (K3468, DAKO)
Counterstaining with hematoxylin

controls

Positive control: Specificity of anti-SOX2 staining using the D6D9 antibody (shown in Supplemental Figure 1)
Negative control: Not explicitly mentioned

Annotations

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