Immpact vector red ap substrate kit

FFPE tissue sections (4-μm thick) were prepared. Antigen retrieval was performed using the Epitope Retrieval Solution pH 9 (Leica Biosystems, Wetzlar, Germany). To block endogenous peroxidase activity, the sections were incubated in a 0.3% H₂O₂ solution in methanol. Ten percent goat serum (Nichirei Bioscience, Tokyo, Japan) was used to block the sections. The primary antibodies used were HPV-L1 antibody (K1H8) (Thermo Fisher Scientific, Waltham, MA, USA), p16 (E4H6) (Roche, Basel, Switzerland), and Ki-67 (MIB-1) (DAKO, Santa Clara, CA, USA). HPV type 6 E4 and type 11 E4 antibodies, as described previously, were also used^{21 (link)}. For secondary antibodies, a MAX-PO(M) or MAX-PO(R) kit (Nichirei Bioscience, Tokyo, Japan) was used. The sections were visualised with DAB.
L1 and E4 double staining was performed by L1 and visualised by DAB, and E4 was visualised using the ImmPACT Vector Red AP Substrate Kit (Vector Laboratories, Newark, CA, USA).
IHC evaluations were performed independently by two pathologists who were blinded to patient information. IHC was evaluated as positive or negative for L1 and E4, the MIB-1 index was calculated as percentage of Ki-67 IHC-positive cells/total tumour cells, and p16 was calculated as the immunoreactive score (IRS) using staining intensity as percentage of the positive tumour cells (Suppl. Fig. S2)^{37 (link)}.

The samples were fixed in 10% neutral buffered formalin and embedded in paraffin wax, which was followed by serial dehydration using ethanol and clearance using xylene. For bone tissues, samples were decalcified with 10% EDTA (pH 7.4) after formalin fixation and delipidation using ethanol. Sections cut into 4-μm thickness were used for Safranin-O/fast green/hematoxylin staining (Saf-O), Alcian blue (pH 1.0) staining (AB), tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry. The information regarding the antibodies and reaction conditions is listed in Additional file 2. After reaction with HPR or AP conjugated antibodies, positive signal color was developed with Histofine Simple stain DAB (NICHIREI BIOSCIENCES, Tokyo, Japan), Vina Green Chromogen Kit (BIOCARE MEDICAL, CA, USA), or ImmPACT Vector Red AP Substrate Kit (VECTOR LABORATORIES, CA, USA). Positively stained areas were measured using ImageJ (National Institutes of Health, MD, USA), ImageScope (Leica Microsystems, Wetzlar, Germany), and BZX-700 (Keyence, Osaka, Japan).