Diaminobenzidine kit

Colon samples (5 μm cuts) were deparaffinised and hydrated with PBS. We performed antigenic recovery in citrate buffer (10 mM, 0.05% Tween-20) at 90 °C for 10 min. Then, the colon samples were chilled and washed with citrate buffer for 20 min. Endogenous peroxidase was blocked by inhibition with 3% H₂O₂ in PBS for 30 min. A block with FBS at 3% was made. The samples were incubated with the primary antibody anti-LDH-A peroxidase 1:100 (Genetex, Irvine, CA, USA) overnight at 4 °C in a humid chamber. The diaminobenzidine kit was added at a 1:9 dilution (Vector Laboratory, Newark, CA, USA), subsequently counterstained with haematoxylin (1:9), washed with tap water, and then washed with distilled water. Finally, the samples were dehydrated in alcohol and xylene and analysed with microscopy (Nikon, Eclipse E600, Melville, NY, USA).

Briefly, sections were incubated overnight at 4°C with rabbit DSP antibody (1:200); immunization of rabbit with the synthetic peptides NH2‐GNKSIITKESGKLSGS‐COOH (amino acid residues 372 ~ 387 of DSP) and BSP antibody (1:200) in 5% skim milk/PBS, pH 7.4. Negative control sections were incubated with 5% skim milk/PBS. Sections were then incubated with a biotin‐labeled goat anti‐rabbit immunoglobulin G (IgG) (1:200; Vector Labs) as the secondary antibody and then washed and incubated with avidin‐biotin‐peroxidase complex (Vector Laboratories Inc., Burlingame, USA). Peroxidase was revealed by incubation with methanol containing 3% H₂O₂. Signals were converted using a diaminobenzidine kit (Vector Laboratories). Nuclei were stained with Hematoxylin (Vector Laboratory, Burlingame, CA, USA) (n = 3, each group).

Mandibles were collected from three control littermates and three Runx2 mutant. The dissected mandibles were fixed, decalcified and embedded by paraffin as previously described^{37 (link)}. Sagittal sections of paraffin-embedded mandibular molars were prepared and used for hematoxylin-eosin (H&E) staining or IHC. For IHC analysis, sections were incubated with rabbit polyclonal primary antibodies, including anti-Runx2 (1:50 dilution; ab23981; Abcam, Cambridge, MA, USA), anti-AMELX (1:1,200 dilution; ABT260; Merck KGaA, Darmstadt, Germany), and anti-KLK4 (1:500 dilution; ab71234; Abcam, Cambridge, MA, USA), at 4 °C overnight. Bound antibodies were detected using a Vectastain ABC Elite Kit and diaminobenzidine kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Subsequently, the tissue sections were counterstained with Mayer’s hematoxylin and examined by light microscopy (BX53F; Olympus. Tokyo, Japan). Each set of experiments was repeated at least three times.

All incubations occurred at room temperature. Prior to immunohistochemical labeling, human tissues underwent antigen retrieval by incubating sections for 10 minutes in a solution of Tris-buffered saline (TBS) containing 20 μg/ml proteinase K, followed by a 10-minute incubation in distilled water containing 3% H₂O₂. Sections were then pre-incubated for 24 h in TBS + 0.05% tween containing 2% normal goat serum, before being transferred for 48 hours into the same solution containing polyclonal rabbit anti-IBA1 (1:1000; WAKO Chemicals USA, Inc., Richmond, VA, USA). This was followed by 1 hour incubation in biotinylated goat anti-rabbit antibody (1:1,000; Vector Laboratories Inc., Burlington, ON, Canada), and the avidin-biotin complex procedure (ABC Kit, Vectastain Elite, Vector Laboratories Inc., Burlington, ON, Canada) for 30 minutes. Labeling was revealed with a diaminobenzidine kit (Vector Laboratories Inc., Burlington, ON, Canada) and samples were counterstained with cresyl violet to better differentiate gray and white matter (Figure 1). Sections were mounted on glass slides, dehydrated, and coverslipped with Permount (Fisher Scientific Inc., Pittsburgh, PA, USA). Mouse brain sections underwent the same procedures, with the exception of the antigen retrieval step.

Cryostat sections at 60 μm thickness were collected and kept free floating at -20 °C in cryopreservative medium until immunolabeling was performed. The sections were washed several times in 1 M PBS, pH= 7.4, then soaked for 1 h in 10% normal donkey serum (Dutscher, Brumath, France) in PBS containing 0.5% Triton X-100 and 0.2% merthiolate. Sections were incubated overnight at room temperature with a goat polyclonal antibody against ChAT (1:200, Chemicon International, Temecula, CA), then incubated for 1 h with biotinylated secondary antibody with peroxidase against goat IgG (1:200; Chemicon International, Temecula, CA). Sections were soaked for 1 h in ABC Vectastain Elite kit (1:500; Vector Labs, Burlingame, CA) before using a diaminobenzidine kit (Vector Labs, Burlingame, CA) to reveal positive neurons. Brain sections were then mounted onto gelatin-coated slides, dried, dehydrated, and cover-slipped. Omission of the primary antibody served as the negative control and resulted in no detectable staining.