3 3 diaminobenzidine dab substrate kit

An immunohistochemical examination was carried out to analyze the biological characteristics of the cells in the dental pulp tissues cultured using the improved CMDPT. The sections were deparaffinized with xylene, rehydrated in descending concentrations of ethanol, and washed in PBS. After antigen retrieval in a 0.01 mol/L sodium-citrate-buffered solution (pH 6.0) at 98 °C for 45 min, the sections were cooled and washed well with PBS. Endogenous peroxidase was blocked by treatment with 3% H₂O₂ in methanol for 1 h at room temperature (RT). After treatment with 10% normal goat serum at RT for 10 min, the sections were incubated with rabbit anti-Ki-67 antibody (Abcam, Cambridge, UK) (diluted 1:200 in PBS containing 1% bovine serum albumin) or rabbit anti-stromal cell-derived factor-1(SDF1) antibody (Abcam, Cambridge, UK) (diluted 1:1000 in PBS containing 1% bovine serum albumin) at 4 °C overnight. After washing with PBS, the localization of each molecule was determined using a Histofine SAB-PO (R) kit (Nichirei Biosciences, Inc., Tokyo, Japan) and 3, 3′-diaminobenzidine (DAB) substrate kit (Nichirei Biosciences, Inc.). The sections were counterstained with hematoxylin and mounted. The specificity of the immunoreaction was confirmed by incubation with normal rabbit IgG instead of primary antibody.

Sections from the left lateral lobe and caudate of the liver were subjected to immunohistochemistry (IHC) with primary antibodies as listed in Supplemental Table S2. After dewaxing and antigen retrieval, tissue sections were immunostained in a Histostainer system (Nichirei Biosciences, Tokyo, Japan) as described previously^{71 (link)}. Briefly, sections were treated with 5% skimmed milk in phosphate buffered saline (PBS) for 10 min, with each primary antibody at room temperature for 1 h, with 3% H₂O₂ in PBS for 15 min, and with horseradish peroxidase-conjugated secondary antibody (Histofine Simple Stain MAX PO; Nichirei Biosciences) at room temperature for 30 min. Positive reactions were visualized with 3,3′-diaminobenzidine (DAB substrate kit; Nichirei Biosciences). After immunohistochemistry, sections were stained with Perls solution for detection of tissue iron.

Sections of human buccal mucosa and rabbit gingiva were transferred onto poly-l-lysine-coated glass slides (Matsunami Glass, Osaka, Japan). After deparaffinization with xylene and rehydration with descending concentrations of ethanol, endogenous peroxidase was blocked by treatment with 3% H₂O₂ in methanol for 1 hour at room temperature (RT). After treatment with 10% normal rabbit serum at RT for 10 min, sections were incubated with the primary antibodies (goat polyclonal antibody against Ob-R, Santa Cruz Biotechnology, Inc., CA, USA) diluted 1∶500 in PBS (pH 7.4) containing 1% bovine serum albumin at 4°C overnight. After washing with PBS, the localization of Ob-R was visualized using a Histofine SAB-PO (G) kit (Nichirei Corporation, Tokyo, Japan) and a 3,3′-diaminobenzidine (DAB) substrate kit (Nichirei). Sections were counterstained with hematoxylin and mounted. The specificity of the immunoreaction was confirmed by incubation with normal goat IgG and normal goat serum instead of the primary antibodies.