Vectastain universal quick kit

Tissues in paraffin block were sectioned at 4μm thickness and were processed for immunostaining. Sections were incubated with primary antibodies diluted (1:100) in 2.5% normal horse serum (NHS; Vector Labs) overnight at 4°C, then stained with biotin-conjugated secondary antibodies followed by incubation with peroxidase-streptavidin complex using Vectastain^® Universal Quick Kit (Vector Labs). Immunostaining was performed using 3, 3’-diaminobenzidine (DAB; Sigma) according to the manufacturer’s instructions. Sections were counterstained with haematoxylin for 1 min, mounted, and coverslip was permanently added for light microscopy. IHC scoring was done by using IHC profiler, an open source plugin, as described by Varghese et al. (2014) [17 (link)]. The score was assigned in a four tier system (0–3) viz. high positive (3+), positive (2+), low positive (1+) and negative (0).

To investigate the production of organic matrix by cells grown on scaffolds, an immunohistochemical staining for Type I Collagen (COL1A1) (MILLIPORE, Temecula, CA, USA) was performed. After endogenous peroxidase blocking and antigen unmasking, sections were incubated with primary mouse monoclonal anti-human antibodies overnight at 4 °C. Secondary antibodies (Vectastain Universal Quick kit; Vector Laboratories, Burlingame, CA, USA) and a development kit (Vector Nova Red; Vector Laboratories) were applied following the manufacturer’s instructions. Representative images were captured with an optical microscope (BX51, Olympus Italia) connected to an XC50 Olympus digital camera (Olympus Italia) and to an image analyzer system (CellSens Dimension, Life Sciences Imaging Software, Olympus Italia).

The maxillary tissues were fixed with 10% formalin (Sigma Aldrich, USA), decalcified with 0.5 M ethylene-diamine tetra acetic acid (EDTA, pH 7.4), and paraffin-embedded. The specimens were sliced into 5 µm thick sections, and TRAP and hematoxylin-and-eosin (H&E) staining analyses were performed, as previously mentioned [2 (link)]. TRAP-positive cells were counted using Image J (Media Cybernetics, Inc. Rockville, MD, USA) [33 (link)].
To identify proinflammatory cytokine expressions, IHC analysis was performed with primary antibodies against IL-1β (AF-401-NA, R&D Systems, Minneapolis, MN, USA), IL-6 (ab6672, Abcam, UK), and TNF-a (sc-133192, Santa Cruz, CA, USA). For the secondary antibody reactions, VECTASTAIN^® Universal Quick Kit (PK-7800, Vector Laboratories, Burlingame, CA, USA) for IL-1β and Dako EnVision+ System Kit (K4009, Dako, Denmark) for IL-6 and TNF-a were used. The DAB Peroxidase Substrate Kit was used to visualize the cytokine expressions in the tissues [2 (link)]. The quantitative comparisons were conducted by calculating the percentage of positive staining on the same-sized area in the furcation of the maxillary second molar using Image J Fiji (Version 1.2).

The ileum was open longitudinally and flushed by cold PBS until most of the luminal content were clear. The tissues were fixed in 4% paraformaldehyde overnight and embeded in paraffin wax. Then the tissues were sectioned (3–4 μm slices). After dewaxing and hydration, antigen retrieval was performed by boiling slices for 30 min in 10mM sodium citrate buffer pH6.0. Samples were blocked with 3% BSA (IHC grade) and incubated with primary antibody at 4 °C overnight, Antibodies used were rabbit anti-ki67(1:200, cell signaling), mouse anti-BrdU (1:400, cell signaling), goat anti-oxytocin receptor (1:100, Abcam) rabbit anti-lysozyme (1:200, DAKO). For immunohistochemistry, the VECTASTAIN Universal Quick Kit, R.T.U. (Ready-to-Use) kit (vectorlabs) was used as secondary regents. Stainings were developed with DAB. Slides were counterstained with haematoxylin and mounted for observation under a Nikon eclipse 80i light microscope. The immunohitochemistrical staining data were analysed double blindly using image-pro plus software. For immunofluorescent, Alexa Fluor® 568 donkey anti- rabbit and Alexa Fluor® 488 donkey anti-goat (life technologies) were used as secondary antibodies. Then sections were counterstained with DAPI. All slides were imaged with a Zeiss LSM780 laser scaning confocal microscope.