Dako lsab system hrp kit

Human gingival tissues were obtained from five healthy volunteers (three male, two female; mean age = 46.7), and from five adult chronic periodontitis patients (three male, two female; mean age = 49.2) at Department of Periodontology, Kagoshima University, Kagoshima, Japan. Tissues were immunostained using anti-K6F antibody according to the method described in our previous study [23 (link)]. Half of the excised tissues were fixed in formalin, incubated in 20% sucrose solution, embedded in OCT and then stored at −80°C until use. The other half of the tissue samples were used to obtain membrane proteins. Staining of frozen tissue sections (4-μm thick) was carried out using the indirect immunoperoxidase diaminobenzidine (DAB) method with a DAKO LSAB+ System HRP kit (KO679; DakoCytomation, Carpinteria, CA, USA).

For immunohistochemical staining Dako LSAB+System-HRP kit was used (Dako North America, California, USA). For the examination for mesenchymal markers, 2×10⁴ cells/well of each sample were seeded on a glass slide (20 mm) in a 12-well plate (Greiner Bio-One GmbH, Frickenhausen, Germany) and then fixed with 4% PFA. After washing steps with PBS, six drops of 3% hydrogen peroxide were added to each well for 5 min to block endogenous peroxidase activities. The cells were incubated with appropriate antibody over night at 4°C (Table S1). Then the link solution was applied to the glass slides for 30 min followed by streptavidin peroxidase for 30 min. The substrate solution was added to each well and incubated for 7–15 min until the antibody horseradish peroxidase conjugate (AB-HRP) catalyzed the 3,3′-diaminobenzidine (DAB) substrate into the desired brown to black reporter color for a positive signal. Afterwards, cells were washed twice with 1 ml distilled water (dH₂O) and treated with 300 µl haematoxylin for 3–5 min providing counterstaining of the nuclei. The glass slides were additionally rinsed with dH₂O, transferred upside down onto an object plate in one drop of Mowiol 4–88 (Carl Roth GmbH, Karlsruhe, Germany) and dried over night in the dark. Images were taken using a Keyence Biozero microscope (Keyence Germany GmbH, Neu-Isenburg, Germany).

Paraffin-embedded tissue sections were de-paraffinized and rehydrated with graded ethanol. PBS containing 3% H₂O₂ was used to block the endogenous peroxidase activity of the samples. The slides (5 μm thick) were incubated with rabbit anti-Chromogranin A (ChgA) polyclonal antibody (1:200, Abcam, ab45179) overnight at 4°C and probed with secondary antibodies (1:500, Invitrogen) for 60 min. Peroxidase activity was visualized using a Dako LSAB + System-HRP Kit according to the manufacturer’s instructions (Dako Cytomation). Images were captured using a manual inverted microscope Leica DMI3000 B. The ChgA immunostaining densities from five random fields were analyzed.