Cell conditioning solution ultra cc1

To detect COL11A1 expression, IHC was performed on Ventana Discovery Ultra autostainer (Roche, Indianapolis, IN) with the following protocol and reagents. Vial of mAb anticol11A1 (Oncomatryx, High Concentration 2.3 mg/mL Rabbit monoclonal (Clone 1e8.33)). All reagents were provided by Roche, Indianapolis IN: Samples underwent heat-induced epitope retrieval, (Cell Conditioning Solution ULTRA CC1 (950-224, Roche)) for 32 min; the primary antibody was incubated for 30 min per manufacturers instruction; followed by DISCOVERY OmniMap anti-Rt HRP (760-4457; Roche), DISCOVERY ChromoMap DAB kit (760-159; Roche), Hematoxylin II (790-2208; Roche), and Bluing reagent (790-2037; Roche) were used for visualization. All samples (PDX, xenograft, primary, normal) were stained in along with positive control (LM7 xenograft) and negative control xenograft (143B COL11A1 KO xenograft), grown subcutaneously in NSG female mice, and tumors were harvested when they reached a size of 1000 mm³. Isotype controls were used as well.

To detect COL11A1 expression, IHC was performed on Ventana Discovery Ultra autostainer (Roche, Indianapolis, IN) with the following protocol and reagents. Vial of mAb anticol11A1 (Oncomatryx, High Concentration 2.3 mg/mL Rabbit monoclonal (Clone 1e8.33)). All reagents were provided by Roche, Indianapolis IN: Samples underwent heat-induced epitope retrieval, (Cell Conditioning Solution ULTRA CC1 (950–224, Roche)) for 32 minutes; the primary antibody was incubated for 30 minutes per manufacturers instruction; followed by DISCOVERY OmniMap anti-Rt HRP (760–4457; Roche), DISCOVERY ChromoMap DAB kit (760–159; Roche), Hematoxylin II (790–2208; Roche), and Bluing reagent (790–2037; Roche) were used for visualization. All samples (PDX, xenograft, primary, normal) were stained in along with positive control (LM7 xenograft) and negative control xenograft (143B COL11A1 KO xenograft), grown subcutaneously in NSG female mice, and tumors were harvested when they reached a size of 1000 mm³. Isotype controls were used as well.

Formalin-fixed, paraffin-embedded tissue sections were stained on the Ventana BenchMark Ultra automated staining platform (Ventana Medical Systems Inc., Tucson, AZ, United States) as recently described (Mitash et al., 2019 (link)). Slides were pretreated with ULTRA cell conditioning solution CC1 (Ventana) for 64 min and stained using mouse monoclonal primary antibody against LMTK2 (Acris AM20991PU-N), at 1:100 dilution. OptiView 3,3′-diaminobenzidine (DAB) IHC detection kit with OptiView amplification indirect, biotin-free multimer amplification system (Ventana) was used for detection of primary antibodies. All slides were counterstained with hematoxylin and routinely dehydrated, cleared, and coverslipped in resinous mounting media.