Histofine simple stain mouse max po

Xenograft tumors of mice treated with either TW-37 or control were paraffin embedded. All tumors were clinically and pathologically identified as being the primary and only neoplastic lesion. Briefly, 3-μm-thick sections of formalin-fixed paraffin-embedded (FFPE) tumors were deparaffinized, and antigen retrieval was performed by boiling the section in citrate buffer at pH 6 or EDTA at pH 9 for 20 min. As primary antibody Ki67 (mib-1, 1:100, pH 6, Thermo Scientific, Waltham, MA, USA) was used. Corresponding secondary antibody detection kits for reduced background on murine tissue were used (Histofine Simple Stain Mouse MAX PO, medac) and stained on an automated stainer (LabVision Autostainer 480S, Thermo Scientific). For cleaved caspase 3 (Cell Signaling Technologies, Danvers, MA, USA) staining of paraffin sections, antigens were retrieved with EDTA buffer (1 mmol/L EDTA, pH 8.0), peroxidases blocked 10 min in 3% hydrogen peroxide, and the antibodies were diluted in Tris-buffered saline containing 1% bovine serum albumin and 5% normal goat serum 1:200. The histochemistry was performed with Super Sensitive Link Label IHC Detection system (BioGenex, San Ramon, CA, USA) and visualized with diaminobenzidine (DAB; Dako, Giostrup, Denmark).

Briefly, 3-μm-thick sections of formalin-fixed paraffin-embedded tumors and adrenals were deparaffinized, and antigen retrieval was performed by boiling the section in citrate buffer at pH 6 or EDTA at pH 9 for 20 min. Staining was performed as previously described^{31 (link)} using anti-cleaved caspase 3 (#9661, Cell Signaling, 1:200), anti-tyrosine hydroxylase (ab76442; Abcam; 1:200), anti-Ki-67/Mib-1 (RM-9106, Dako Deutschland GmbH, Hamburg, Germany, 1:25) and anti-Ncam1 (ab6123; Abcam, Cambridge, UK; 1:500) as primary antibodies. Corresponding secondary antibody detection kits for reduced background in murine tissues were used (Histofine Simple Stain Mouse MAX PO, Medac, Hamburg, Germany), and antibody complexes were visualized using an automated stainer (LabVision Autostainer 480S, Thermo Scientific, Langenselbold, Germany). All slides were scanned with a Pannoramic 250 slide scanner (3D Histech.com Budapest, Hungary, Electron microscopy was performed as previously described.^{32 (link)}