Novared hrp substrate kit

The processes of tissue preparation, section and staining have been described previously [22 (link)]. The signals were visualized using the NovaRed HRP substrate kit (VECTOR, Burlingame, CA). The sections were counterstained with hematoxylin.
To evaluate the expression levels of NMU and NMUR2 proteins in the endometrial cancer tissue microarray, the averaged staining intensity in each core of the tissue microarray was quantified and scored as 0, 1, 2 or 3. The H score of each core was calculated by multiplying the intensity score by the percentage of positive staining in each core.

The clinical materials of ovarian cancers and the paired adjacent normal tissues were fixed by Bouin’s solution following paraffin embedding. The antigens on 5 μm-thick sections were retrieved by Target Retrieval Solution, pH 9 (DAKO). For the performance of immunohistochmeistry, the rabbit polyclonal anti-NMU antiserum was prepared as previously described [18 (link)] and the rabbit polyclonal anti-NMUR2 antibody (Abcam) was used. The rabbit preimmune serum and the normal IgG (Cell Signaling) at the same concentration were used as negative controls, respectively. The staining of NMU and NMUR2 were performed using the Universal LSAB^TM+ Kit/HRP (DAKO) and NovaRed HRP substrate kit (VECTOR) according to the manufacturers’ instructions.