Journal: Smart Materials in Medicine

Article Title: Eggshell-derived amorphous calcium phosphate: Synthesis, characterization and bio-functions as bone graft materials in novel 3D osteoblastic spheroids model

doi: 10.1016/j.smaim.2023.04.001

Figure Lengend Snippet: Fig. 10. Confocal microscopic inspection of OPN and collagen I expression of MC-3T3-E1 cells in 3D spheroids with ACP particles embedded and 2D culture system, respectively. (A) Left panel: OPN and collagen I expression in different culture media in 3D reconstructed images; Right panel: maximal fluorescent projection of OPN and collagen I in 3D spheroids as shown in left penal; (B) OPN and collagen I expression in different culture media in a 2D culture system. Scale bar ¼ 100 μm.

Article Snippet: In brief, cells and spheroids were cultured for 7 days and then fixed with 4 wt % PFA for 20 min. After washing in PBS thrice, samples were treated with 1% (v/v) TritionX-100 (X100, Sigma-Aldrich, USA) for 20 min, followed by blocking in 1 wt % BSA/PBS solution for 1 h. Then, the samples were incubated in goat-anti-mouse OPN primary antibody (Ab) (AF808, R&D systems, USA) and rabbit-anti-mouse collagen I Ab (ab21286, Abcam, USA) at 4 C overnight.

Techniques: Expressing

Journal: Journal of structural biology

Article Title: Nanostructure of mouse otoconia.

doi: 10.1016/j.jsb.2020.107489

Figure Lengend Snippet: Figure 6. Immunogold labeling of OPN in otoconia. (a, c) In wild-type mice, otoconial voids shown by TEM and SEM, respectively, after immunolabeling for OPN (aqueous procedures dissolve the calcitic otoconia). Inset: Intact wild-type mouse otoconia (without aqueous exposure) shown by SEM. By TEM (b) and SEM (d), immunogold labeling for OPN shows gold particles (arrows) at the surface of otoconial voids. (e, g) In OPN-deficient mice, otoconial voids shown by TEM and SEM, respectively, after immunolabeling for OPN. Inset: Intact otoconia from OPN-deficient mice (without aqueous exposure) shown by SEM. By TEM (f) and SEM (h), as expected in this negative control, immunogold labeling for OPN was absent.

Article Snippet: Otoconia-containing thin sections and block faces were incubated with anti-mouse OPN antibody (R&D Systems Oakville, Canada) for 1 h followed by washing and incubation with protein A-colloidal gold complex (14-nm gold particles, from G. Posthuma, University of Utrecht) for 1 h to detect the immunolabeling reactions.

Techniques: Labeling, Immunolabeling, Negative Control

Journal: Developmental cell

Article Title: Dermal TRPV1 innervations engage a macrophage and fibroblast containing pathway to activate hair growth in mice

doi: 10.1016/j.devcel.2024.05.019

Figure Lengend Snippet: (A) Diagram of experimental design, representative FACS plots and quantification of CD9+CD26+ dermal fibroblasts percentage from Lin- Thy1+SCA1+ dorsal skin cells after three daily CNO intradermal injections from TRPV1 activated mice and their controls (****p<0.0001). (B) UMAP projection demonstrating the clustering of sorted Thy1+SCA1+ fibroblasts based on scRNAseq data, and the overlay of the Hashtag signal differentiating the subcutis and dermal clusters. (C) Normalized fold change (FC) calculation comparing cell numbers in dermal clusters between TRPV1 activated mice and their controls. (D) UMAP projection of sorted fibroblast scRNAseq data demonstrating cell distribution between clusters in the TRPV1 activated sample and control. (E-H) UMAP projection of sorted fibroblasts demonstrating the expression of (E) Col23A1 (F) Prdm1 (G) Crabp1 and (H) Lef1 as well as the list of top five velocity driver genes for cluster #3. (I) UMAP projection demonstrating calculated DP-core enrichment score and (J) Lepr expression. (K) UMAP projection of scRNASeq from Li et al. demonstrating its calculated DP-score (left) and cluster #3 enrichment score (right). (L) UMAP projection of sorted fibroblasts demonstrating the expression of Spp1. (M) Representative FACS histograms of anti-Spp1 staining of Lin- Thy1+SCA-1+ dermal cells (red) or CD9+CD26+ subset (orange) from the back skin of TRPV1 activated mouse. Secondary only staining control in blue. (N) Quantification of Spp1+CD9+CD26+ dermal fibroblasts from TRPV1 activated mice and controls (*p=0.017). (O) Representative images of RNAscope staining for Spp1 (green) and the Lepr (red) on dorsal skin collected from TRPV1 activated mice and controls. Scale bar, 100um. White arrows point out DP; yellow arrows point out Spp1 expression out of DP. DP images scale bar, 25um.

Article Snippet: InVivoMAb Anti-Spp1 neutralizing antibody (100D3) , BioXCell , Cat # #BE0372; RRID: AB_2927509.

Techniques: Control, Expressing, Staining, RNAscope

Journal: Developmental cell

Article Title: Dermal TRPV1 innervations engage a macrophage and fibroblast containing pathway to activate hair growth in mice

doi: 10.1016/j.devcel.2024.05.019

Figure Lengend Snippet: (A) Diagram of experimental design and representative images from shaved TRPV1 activated mice and controls. Mice monitored for hair coat growth for 12 days after the last TRPV1 activation with CNO (postnatal day 63). (B) Enlarged dashed rectangle from panel A depicting hair growth in a TRPV1 activated mouse. (C) HF length quantification of dorsal skin from mice treated as in A. Data combined from 6 to 8 HF per mouse, taken from 6 mice per group (****p<0.0001). (D) Representative immunofluorescent images of dorsal skin from mice treated as in A. Staining with anti-keratin14 (KRT14; green) marking epidermal layer and Ki67 (purple) marking the HF bulb. DAPI stain in blue. Scale bar 100μm. (E) Quantification of normalized Ki67 MFI signal in the peri-follicular area of dorsal skin from mice treated as in A. Data combined from 4 regions of interest (ROI) per mouse taken from 5 TRPV1 activated mice and 9 controls (****p<0.0001). (F) Diagram of experimental design and representative images from TRPV1 activated mice and controls treated with CNO on half of their back skin (marked with dashed rectangle). Anti-Spp1 neutralizing antibody or IgG control were injected intradermally to the same skin area. Data are combined from 4 HFs per mouse collected from 6 controls, 3 TRPV1 activated + IgG treated and 4 TRPV1 activated + anti-Spp1 treated mice (****p<0.0001). (G-H) Percentage of CD9+CD26+ dermal fibroblasts from TRPV1 activated mice and their controls intradermally injected with (G) QWF (SubP antagonist) or vehicle (**p=0.005, ***p=0.0004), (H) CGRP8–37 (CGRP antagonist) or vehicle (***p=0.0001, ****p<0.0001). (I) Diagram of experimental design and representative images from TRPV1 activated mice and controls pre-treated intradermally with CGRP8–37 or vehicle and monitored for hair coat growth for 14 days. Quantification of HF length from 6 to 8 HFs per mouse, taken from 3–6 mice per group (****p<0.0001). (J) Representative immunofluorescent images of dorsal skin from mice treated as in I. Stained with anti-KRT14 (green), Ki67 (pink), and DAPI (blue). Scale bar, 100μm.

Article Snippet: InVivoMAb Anti-Spp1 neutralizing antibody (100D3) , BioXCell , Cat # #BE0372; RRID: AB_2927509.

Techniques: Activation Assay, Staining, Control, Injection

Journal: Developmental cell

Article Title: Dermal TRPV1 innervations engage a macrophage and fibroblast containing pathway to activate hair growth in mice

doi: 10.1016/j.devcel.2024.05.019

Figure Lengend Snippet: (A) Representative immunofluorescent images and quantification of DRGs collected from mice with naïve back skin or 90 minutes after tape stripping. Stained with anti-c-Fos (red), anti-TRPV1 (green) and DAPI (blue). White arrowheads indicate neuron cell bodies that are TRPV1+ only. Data combined from 6–7 fields of view taken from 4 mice per group (****p<0.0001). Scale bar 50μm. (B) Percentage of CD9+CD26+ dermal fibroblasts from naïve back skin and 18 hours after tape stripping 6 or 12 times (***p=0.0004,****p<0.0001). (C) Diagram of experimental design and quantification of CD9+CD26+ dermal cell percentage 18 hours after tape stripping from the back skin of TRPV1Cre DTR+ treated with DTx or TRPV1Cre DTA+ mice and their controls (****p<0.0001). (D) Representative FACS histogram of anti-Spp1 staining. Dorsal skin treated as in C and gated on total Lin- Thy1+SCA-1+ cells (red) and the CD9+CD26+ cell subset (orange). Secondary only staining control in blue. (E) Quantification of Spp1+CD9+CD26+ dermal fibroblasts from TRPV1 ablated and control mice treated as in C (**p=0.005). (F) TRPV1 ablated mice and controls treated as in C and monitored for hair coat recovery for 12 days (postnatal day 61). (G) Representative bright field images of samples described in F. Data for HF length quantification collected from 8 HFs per mouse taken from 6 control and 5 TRPV1 ablated mice (****p<0.0001). (H) Ramp1 KO mice and controls treated as in F. HF length quantification from 4 HFs per mouse taken from 4 control and 5 Ramp1 KO mice (****p<0.0001). (I) Mice treated as in F and intradermally injected with anti-Spp1 or isotype control while being monitored for hair coat recovery for 16 days (postnatal day 65). HF length quantification from 4 HFs per mouse taken from 6 isotype control and 5 anti-Spp1 injected mice (**p=0.005).

Article Snippet: InVivoMAb Anti-Spp1 neutralizing antibody (100D3) , BioXCell , Cat # #BE0372; RRID: AB_2927509.

Techniques: Stripping Membranes, Staining, Control, Injection

Journal: Developmental cell

Article Title: Dermal TRPV1 innervations engage a macrophage and fibroblast containing pathway to activate hair growth in mice

doi: 10.1016/j.devcel.2024.05.019

Figure Lengend Snippet: Key resources table

Article Snippet: InVivoMAb Anti-Spp1 neutralizing antibody (100D3) , BioXCell , Cat # #BE0372; RRID: AB_2927509.

Techniques: Recombinant, RNAscope, Multiplex Assay, In Situ, Software