Ventana ultra

Spheroids were collected and pooled, washed in PBS, and fixed in formalin for 1 h at room temperature. After washing twice in PBS, spheroids were transferred to a cryomold and suspended in Histogel, before paraffin embedding and sectioning (4 μm). Cellular morphology was assessed via hematoxylin and eosin (H+E) staining, and immunohistochemistry was performed using the Ventana Ultra and associated reagents from Roche. Details of the conditions for each of the antibodies used are reported in Table 3.Table 3

Summary of antibodies and conditions used for immunohistochemistry on the Ventana Ultra

Antibody	Supplier	Host	Pretreatment	Dilution
CD68	Novocastra	Mouse	mCC1, pH8	1:100
α-SMA	Dako	Mouse	mCC1, pH8	1:100
COL1A1	Biosite	Rabbit	stdCC1, pH8	1:1000

Diagnostic neuroblastoma samples from the Children’s Oncology Group (COG) and Stanford University, School of Medicine, Department of Pathology were used. After pathology review according to the protocol of the COG Neuroblastoma Biology Study, unused sections were available for immunostaining. Those cases were filed at the COG Neuroblastoma Pathology Reference Laboratory, which were identified only by the COG accession number and not associated with patient information. IRB approval of the cases was obtained at the time of study enrollment by the contributing institution. Neuroblastoma specimens archived at Stanford University were used to perform IHC staining on serial sections of the tumor tissues. Formalin-fixed paraffin embedded tumor sections were subjected to IHC analysis. Clone KP1 for CD68 (Dako) and clone MRQ-26 for CD163 (Ventana/Cell Marque) were used to stain the corresponding antigens. The automated IHC processor was used to process tumor sections according to the manufacturer’s instructions (Ventana Ultra, Roche). Immunohistochemical evaluation was performed in the representative and viable tumor areas away from necrosis and fibrosis.

Automated immunostaining was performed on the Ventana Ultra (Roche). The protocol involved dewaxing with Ezprep solution, followed by heat-induced epitope retrieval with CC1buffer for 64 min and then endogenous peroxidase inhibition. Slides were then incubated with the ready to use BRAF V600E mutation-specific monoclonal antibody, (Ventana, clone VE1 (CE-IVD)) for 16 min at 37 °C. Chromogenic detection was carried out using the OptiView DAB IHC kit (Roche) along with an Optiview amplification kit (Roche). Four minutes Optivew amplification H2O2 and four minutes Optiview amplification multimer incubation times were used. Slides were counterstained with haematoxylin II (Roche) for 4 min followed by bluing reagent (Roche) for 4 min.
Positive staining was seen as the presence of unequivocal cytoplasmic granular staining of any intensity in the tumour cells. Negative staining showed the absence of cytoplasmic staining in the tumour cells.