Envision kit hrp dab

Immunohistochemical analysis (IHC) of LAT1 was performed using surgically resected intracranial tumor tissue. For immunohistochemical analysis, a rabbit anti-canine LAT polyclonal antibody was used. This antibody was prepared using a synthetic peptide antigen designed based on the C-terminal amino acid sequence of canine LAT1 [19 (link)]. Immunohistochemical analysis was performed using the following method: After deparaffinization, the sections were microwaved in 0.01 M citric acid (pH 6.0) for 3 min, heated five times, and washed with 0.01 M phosphate-buffered saline (pH7.4). Endogenous peroxidase was inactivated with methanol containing 0.3% H₂O₂, and the sections were incubated in rabbit anti-dog LAT1 polyclonal antibody at 4°C overnight. Immunostaining was performed using a commercially available kit [EnVision + kit/ HRP (DAB), Dako, Glostrup, Denmark). Subsequently, 3,3′-diaminobenzidine (DAB) H₂O₂ solution was applied to induce a positive color reaction. After the DAB reaction, specimens were washed three to four times with deionized water, and nuclei were stained with hematoxylin for observation.

For preparation of the paraffin block of 293T cells, the cells were fixed in 20% of formalin/PBS for 24 h. After removing the formalin, alcohol dehydration and paraffin permeation were done using Tissue-Tek VIP5Jr (Sakura, Alphen aan den Rijn, The Netherlands). Paraffin blocks were sectioned at 3-μm thickness. The sections were then transferred to coating slide glasses (Muto pure chemicals, Bunkyo-ku, Tokyo, Japan). After paraffin removal, the paraffin sections of the 293T and ATL cells were treated with 3% H₂O₂. Antigen-retrieval treatment was done using Histofine antigen retrieval solution pH9 (Nichirei, Chuo-Ku, Tokyo, Japan) for 20 min under microwave radiation. After reaction with the first antibody, anti-EVC antibody (HPA008703, 1:400; SIGMA), and the second antibody (K5027, ENVISION Kit/HRP [DAB]; Dako, Bunkyo-ku, Tokyo, Japan), the sections were colored using ENVISION Kit/HRP [DAB] DAB+ (K3468; DAKO). Finally, the sections were stained with hematoxylin.