Vectastain elite abc rabbit immunoglobulin g kit

The expression levels of ARC proteins were evaluated by IHC. All specimens were fixed in 10% buffered formalin and embedded in paraffin. The 3.5 μm thick sections were subjected to antigen retrieval for 20 min at 110 °C in 10 mM citrate buffer at pH 6.0, and the endogenous peroxidase activity was blocked with methanol supplemented with hydrogen peroxide. Sections were blocked by goat serum, incubated with the anti-ARC rabbit polyclonal antibody (16290-1-AP, Proteintech) at a dilution of 1:200 and anti-E-cadherin monoclonal rabbit antibody (#3195; CST) at dilution of 1:400, overnight at 4 °C, and then incubated with the secondary antibody at a 1:200 dilution at 25 °C for 30 min using VECTASTAIN Elite ABC Rabbit Immunoglobulin G kit (Vector Laboratories, Burlingame, CA, USA). We used human brain tissue as a positive control and assigned the specimen with the same intensity of staining as the positive control to the ARC strong group, whereas the unstained specimen was assigned to the ARC negative group. We assigned the specimen stained weaker than the positive control to the ARC weak positive group (Supplementary Fig. 10a).

With a written informed consent, tissue samples were collected from the HCC cases who were diagnosed with imaging studies including magnetic resonance imaging and computed tomography, and underwent surgical resection in Niigata University Hospital. The tissues were stained with hematoxylin and eosin or immunohistochemical stains: Rat anti‐Ctip2 (Bcl11b) antibody (ab18465; Abcam), Rabbit anti‐GATA6 antibody (ab175349; Abcam), Vectastain Elite ABC Rat immunoglobulin G kit (PK‐6104; Vector Laboratories), Vectastain Elite ABC Rabbit immunoglobulin G kit (PK‐6101; Vector Laboratories, Burlingame, CA), and 3,3′‐diaminobenzidine chromogen tablets (Muto Pure Chemicals). The expression of BCL11B and GATA6 was confirmed via RT‐PCR in recently resected 70 cases using the aforementioned procedure. The tumor tissues of these cases were immunohistochemically stained to determine the relationship between BCL11B and GATA6. Images were captured randomly, and analyzed quantitatively, using the ImageJ software (version 1.8.0_172; National Institutes of Health, Bethesda, MD).³⁸