Anti his tag primary antibody

The coding sequence of CrSS was amplified with primers oxSS-F and oxSS-R (Supplemental Table S1), which introduce an AgeI restriction site in both extremities. The PCR product was digested and ligated in an AgeI/XmaI-digested pEAQ-HT plasmid in order to generate the pEAQ-HT:CrSS-6HIS construct. The plasmid was electroporated in A. tumefaciens GV3101, and cells were prepared for infiltrations as described in the section on VIGS methodology. Agrobacteria harboring either an EV (pEAQ-HT) or the pEAQ-HT:CrSS-6HIS (oxCrSS) vector were mixed with Agrobacteria harboring a GFP-expressing vector (pEAQ-HT:GFP-6HIS) in a final OD₆₀₀ = 0.25 each. Catharanthus roseus plants (cv Little Bright Eyes) were vacuum infiltrated and leaf areas exhibiting GFP fluorescence were collected at 6 d post infiltration for further analyses. Proteins were extracted as described previously (Carqueijeiro et al., 2020 ) and resolved on 8% SDS–PAGE, followed by Coomassie Brilliant Blue staining or western blotting. Western blot analysis was performed with an anti-HIS tag primary antibody (#ab213204, Abcam) and a horseradish peroxidase-conjugated secondary antibody (#1706515, Bio-Rad) following standard protocols. The alkaloid content of agroinfiltrated leaves was determined using UPLC-MS analysis as described previously (Dugé de Bernonville et al., 2017 (link)).

We used longitudinal tibia sections for TUNEL and immunostaining. We deparaffinized, rehydrated and incubated the sections in citric acid buffer (10 mmol·L^–1, pH 3) for 60 min at 37 °C (Vector Labs, Burlingame, CA) for antigen retrieval, and 20 min in 1X animal-free blocker (Vector Labs, Burlingame, CA) prior to specific stainings. For detection of endogenous DMP1 in cortical bone, we incubated the sections with anti-DMP1 primary antibody (#ab103203, C-terminal region, Abcam, Cambridge, MA, USA) for 1 h. For detection of injected His-tagged recombinant DMP1 in cortical bone, we incubated sections with anti-His tag primary antibody (Abcam, Cambridge, MA) for 1 h. We then used the immunohistological Vectastain ABC kit (Vector Labs, Burlingame, CA) and performed detection by bright-field microscopy (Leica Microsystems, Buffalo Grove, IL, USA). We performed TUNEL staining using ApopTag Peroxidase In Situ Apoptosis Detection Kit according to manufacturer’s protocol (Millipore Corporation, Temecula, CA) and quantified the ratio of TUNEL-positive osteocytes to total osteocytes on three separate sections per animal.