Discovery staining module

Tumor sections (5 μm) were cut from paraffin-embedded tissue, mounted on Fisherbrand Superfrost/plus slides (ThermoFisher Scientific) and stained with the rabbit polyclonal claudin 1 antibody (Life Technologies) at a dilution of 1:150. This antibody has been previously validated for specificity and sensitivity in our laboratory [18 (link)]. IHC was performed as recently reported [25 ] using an automated tissue immunostainer (Discovery Staining Module, Ventana Medical Systems, AZ, USA). Briefly, sections were dewaxed in two xylene baths, taken through a series of alcohols, rehydrated and then submitted to heat-induced antigen retrieval for 8 min in the presence of a citrate buffer (CC1, Ventana Medical Systems) using the automated tissue immunostainer (Discovery Staining Module, Ventana Medical Systems). Primary antibodies were applied for 60 min and secondary antibodies for 32 min.
Positive staining for claudin 1 protein was assessed using semi-quantitative scoring (H-scores). H-scores were derived from both staining intensity (scale 0–3) and percentage of positive cells (0–100%), which when multiplied, generated a score ranging from 0–300. Tissue microarray (TMA) staining was evaluated independently by two investigators (AB, CP) and where discordant, cases were re-evaluated jointly.

IHC for TMAs was performed as described previously [18 (link)]. Serial sections (approximately 5 μm) were stained with antibodies as previously described [5 (link)]. Briefly, sections were submitted to antigen retrieval (CC1, Ventana Medical Systems, Tucson, AZ, USA) using an auto-immunostainer (Discovery Staining Module, Ventana Medical Systems), followed by one-hour incubation with primary antibody and 32-minute incubation with secondary antibody. Primary antibody concentrations initially applied to the Ventana instrument were 1:50 for antibodies to p-S2448 mTOR, mTOR, p70S6K and p-T389 p70S6K translating into final concentrations of 1:150 after 1:3 dilution with buffer dispensed onto the slide with the primary antibody.