Dab substrate kit

For whole mount staining, E15.5 and E18.5 embryos were harvested, skinned, eviscerated, and fixed in 95% ethanol overnight followed by 95% acetone overnight. Specimens were stained with Alcian Blue for cartilage (0.03% in acetic acid/95% ethanol—1:5 ratio), cleared in 1% KOH, and subsequently stained in Alizarin Red for bone (0.01% in KOH).
For immunohistochemistry staining, tissue was harvested, fixed in formalin, decalcified in 12% ethylenediaminetetraacetic acid (EDTA) (pH 7.4), and paraffin‐embedded. Histological sections were cut 4‐μm thick and paraffin was removed in xylene, followed by rehydration with a graded ethanol series (100%, 95%, 90%, and 70%) for 3 min each. Specimens were treated with proteinase K for 15 min for antigen retrieval and incubated with 3% H₂O₂ for 15 min to quench endogenous peroxidase activity. Specimens were then blocked for 30 min using 1% BSA blocking buffer (Sigma‐Aldrich). Primary antibodies against mouse Metrnl and isotype controls including rat immunoglobulin G (all antibodies where purchased from R&D Systems) were applied as per manufacturer's guidelines and incubated at 37°C for 2 h. This process was followed by incubation with a corresponding anti‐rat secondary antibody for 30 min at 37°C. Specimens were developed using DAB Substrate Kit (Vector Laboratories) and counterstained using Mayer's hematoxylin (Sigma‐Aldrich).

Immunohistochemical staining was performed to detect the accumulation of the CYP1A1 protein in the palatal sections of mice at the GD16 stage. Antigen activation was performed using an Immunosaver (Nisshin-EM Co., Tokyo, Japan) at 98 °C for 45 min. To avoid nonspecific background staining, tissue sections were blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at room temperature. Sections were stained with CYP1A1 polyclonal antibody (13241-1-AP, Cosmobio, Tokyo, Japan; dilution 1:100) and IgG1 Isotype Control (MAB002, R&D Systems, MN, USA; dilution 1:200). The cells were stained at 4 °C overnight. The sections were then incubated with biotinylated secondary antibodies (Iwai Chemicals Co., Tokyo, Japan) for 2 h at room temperature. After incubation, streptavidin-horseradish peroxidase (BioLegend, San Diego, CA, USA) was applied for 60 min. The DAB Substrate Kit (Vector Laboratories, Burlingame, CA, USA) was used for color development according to the manufacturer’s instructions. The sections were then contrast-stained with hematoxylin. Immunohistochemical staining of sections of the palate was performed using a UPM Axio Phot2 (Carl-Zeiss).

Tissue specimens were processed and stained as previously described^{1 (link)}. Slides were stained with primary antibodies (Phospho-Paxillin (Tyr118), 1:100, Cell Signaling; ACP6, 1:50, Abcam; p53 (Pantropic), 1:100, Millipore Sigma; Ki-67, 1:200, Thermo Fisher Scientific) in 1.25% normal horse serum/PBS or 20% goat serum/TBST overnight at 4 °C. For IHC, slides were visualized using VECTASTAIN Elite ABC HRP kit and DAB Substrate Kit (Vector Laboratories) and counterstained with hematoxylin (Thermo Fisher Scientific). For IF, slides were probed with fluorescent secondary antibodies (1:200, Thermo Fisher Scientific) and Hoechst 33258 (1:200, Thermo Fisher Scientific). Images were acquired with a Nikon Eclipse Ti2 microscope and images processed and quantified with Fiji-ImageJ or NIS-Elements HC.