To prepare samples for IHC, extracted teeth were immediately fixed in Karnovsky solution for 48–72 h at 4ºC and then underwent decalcification in 10% EDTA (pH 7.4) with three changes per week for a total of 8 months. Teeth were paraffin embedded and 6 μm-thick serial sections were obtained. Selected target proteins identified by proteomic analysis were confirmed by IHC in histological sections of primary and secondary teeth (n=3/group) as previously described.28 (link) Briefly, IHC was performed on deparaffinized sections using an avidin-biotinylated peroxidase enzyme complex (ABC)-based kit (Vector Labs, Burlingame, CA, USA) with 3-amino-9-ethylcarbazole (AEC) chromogenic substrate (Vector Labs). Primary antibodies included: Rabbit polyclonal IgG anti-decorin (DCN; LF-114, provided by Dr. Larry Fisher, National Institute of Dental and Craniofacial Research, Bethesda, MD, USA); rabbit polyclonal IgG anti-osteocalcin (BGLAP; Millipore, Burlington, MA, USA); and goat polyclonal IgG anti-myeloperoxidase (MPO; R&D Systems, Minneapolis, MN, USA). Negative controls lacking primary antibody were performed.
3 amino 9 ethylcarbazole aec chromogenic substrate
Manufactured by Vector Laboratories
Sourced in United States
3-amino-9-ethylcarbazole (AEC) is a chromogenic substrate used in immunohistochemistry and enzyme-linked immunosorbent assays (ELISA). It produces a red-brown colored precipitate upon reaction with the enzyme horseradish peroxidase (HRP), which is commonly used as a reporter enzyme in these techniques.
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2 protocols using 3 amino 9 ethylcarbazole aec chromogenic substrate
1
Immunohistochemical Analysis of Dental Tissues
2
Immunohistochemical Validation of Proteomic Targets
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Three proteins identified in the PDL by proteomic analysis were selected. Their presence was confirmed by IHC using additional histological sections from the same animals assessed by LC-MS/MS. IHC was performed on paraffin sections using an avidin-biotinylated peroxidase enzyme complex- (ABC) based kit (Vector Labs, Burlingame, CA, USA) with 3-amino-9-ethylcarbazole (AEC) chromogenic substrate (Vector Labs, Burlingame, CA, USA), as described previously [63 (link)]. The primary antibodies included were Prelp (bs13707R, Bioss, Hong Kong, China), Sec13 (ORB312990, Biorbyt, Cambridge, UK), and Sod2 (PA530604, Invitrogen). Samples were stained with 3,3′-diaminobenzidine (DAB, Dako, Glostrup, Denmark) and counterstained with Carazzi’s hematoxylin. Negative controls lacking primary antibodies were performed. Representative photomicrographs of each sample’s mesial, distal, and furcation areas per group were taken at 400x magnification using a digital camera (DP-71, Olympus, Hong Kong, China) attached to a light microscope (BX-51, Olympus). The analysis was performed by an experienced examiner blinded to the experimental groups by counting positive cells.
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