Journal: Cell Death & Disease

Article Title: Palmitoylethanolamide controls reactive gliosis and exerts neuroprotective functions in a rat model of Alzheimer's disease

doi: 10.1038/cddis.2014.376

Figure Lengend Snippet: Effect of PEA on A β -induced S100B protein transcription, expression, and release. ( a – e ) Representative fluorescent photomicrographs ( × 20 magnification) of S100B immunostaining (red) in the CA3 region of hippocampi ipsilateral to vehicle- or A β 1–42 injection site obtained from rats i.p. treated with vehicle, PEA (10 mg/kg), GW6471 (2 mg/kg), or both drugs. Nuclei were stained with DAPI (blue). ( f ) Data obtained by the semiquantitative analysis of OD of S100B immunostaining. Data are expressed in arbitrary units as means±S.E.M. (*** P< 0.001; ** P< 0.01). ( g ) Results of S100B RT-PCR amplification and ( h ) densitometric analysis of corresponding bands. Data were generated normalizing to GAPDH (** P< 0.01; * P< 0.05). ( i ) Representative western blots for S100B protein from hippocampi ipsilateral to the injection site. ( j ) Densitometric analyses normalized to β -actin loading controls (** P< 0.01; * P< 0.05). ( k ) Measurement of S100B release by ELISA in homogenate hippocampi ipsilateral to theA β injection site (*** P< 0.001; ** P< 0.01). Statistical analysis was performed by one-way ( f ) or two-way ( h , j , and k ) ANOVA followed by Bonferroni multiple comparison test. Results presented as means ±S.E.M. of five experiments

Article Snippet: After quenching auto-fluorescence with 0.05 M ammonium chloride, and saturation of non-specific sites with 3% normal donkey serum (BioCell Research Laboratories, Newport Beach, CA, USA), sections were incubated overnight at 4 °C with 0.5% albumin bovine serum/0.25% Triton-TBS solution containing one of the following primary antibodies: rabbit anti-GFAP (1 : 500; Abcam plc), rabbit anti-S100B (1 : 1000, BD Transduction Laboratories, San Jose, CA, USA), mouse anti-MAP-2 (1 : 250, Novus Biologicals).

Techniques: Expressing, Immunostaining, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Generated, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

doi: 10.1038/s41598-017-12880-9

Figure Lengend Snippet: Blocking S100B early after acute muscle injury delays regeneration. ( a ) BaCl 2 -injured TA muscles were injected with IgG or a polyclonal anti-S100B antibody (Abcam No. ab41548) at d1 p.i. Treated muscles were excised at d3, d7 or d14 p.i. ( b ) Histology of muscles and counts of interstitial cells and centrally nucleated myofibers. ( c ) Counts of PAX7 + , MyoD + , myogenin + , Ki67 + , and RAGE + cells (also see Fig. ). ( d ) Western blots of the indicated proteins in homogenates of IgG- and anti-S100B-treated muscles. Immunoblots of GAPDH and α-actinin are included as loading controls. ( e ) Myofiber size distribution at d14 p.i. in uninjured muscles and IgG- and anti-S100B-treated injured muscles. ( f ) Migration of primary mouse myoblasts in Boyden chambers in the absence or presence of S100B. Full-length blots are presented in Supplementary Fig. “Fig. 1”. Results are means ± SEM (n = 6). ** p < 0.01, *** p < 0.001 vs. control. The scale bar represents 50 µm in ( b ) and 200 µm in ( f ).

Article Snippet: At d1 or d4 p.i., TA muscles were injected with either a neutralizing rabbit polyclonal anti-S100B antibody (Abcam No. ab41548) or rabbit non-immune IgG (Sigma) (100 ng/muscle).

Techniques: Blocking Assay, Muscles, Injection, Western Blot, Migration, Control

Journal: Scientific Reports

Article Title: Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

doi: 10.1038/s41598-017-12880-9

Figure Lengend Snippet: S100B affects macrophages early after acute muscle injury. ( a ) TA muscles were treated as described in the legend to Fig. . Muscles were excised at d3 or d7 p.i. ( b ) Counts of MAC3 + cells. ( c – f,h ) Macrophages were isolated from IgG- and anti-S100B-treated injured muscles and either counted ( c ), analyzed by real-time PCR ( d , f , h ), or subjected to western blotting ( e ) (also see Fig. ). ( g ) Peritoneal macrophages were subjected to a migration assay using Boyden chambers in the presence of increasing S100B doses. ( i ) IgG- and anti-S100B-treated injured muscles excised at d7 p.i. and subjected to collagen IV detection by immunohistochemistry and western blotting. ( j ) Macrophages were isolated from IgG- and anti-S100B-treated injured muscles at d3 p.i., cultured for 24 h in the absence or presence of 200 ng S100B/ml, and analyzed by real-time PCR. ( k ) Macrophages were treated with IFN-γ, IL-10 or IL-4 in the absence or presence of the NF-κB inhibitor, Bay11-7085, and analyzed by real-time PCR for S100b levels. ( l ) Western blot analysis of S100B in conditioned media of IFN-γ-, IL-10- or IL-4-stimulated peritoneal macrophages. ( m ) Proliferation assay of C2C12 myoblasts cultured in the presence of IgG- or anti-S100B-treated conditioned media from vehicle- or IFN-γ-stimulated peritoneal macrophages (left) and differentiation assay of C2C12 myoblasts cultured in the presence of IgG- or anti-S100B-treated conditioned media from vehicle- or IL-10-stimulated peritoneal macrophages (right). Immunoblots of α-tubulin are included as loading controls in western blots in e , m . Full-length blots are presented in Supplementary Fig. “Fig. 2”. Results are means ± SEM (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. # p < 0.01, ## p < 0.001 (d7 vs. d3 p.i., b and c; anti-S100B antibody- vs. IgG-treated, j; BAY11-7085-treated vs. control, k; IFN-γ-treated or IL-10-treated vs. control, m). The scale bar in i represents 50 µm.

Article Snippet: At d1 or d4 p.i., TA muscles were injected with either a neutralizing rabbit polyclonal anti-S100B antibody (Abcam No. ab41548) or rabbit non-immune IgG (Sigma) (100 ng/muscle).

Techniques: Muscles, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Migration, Immunohistochemistry, Cell Culture, Proliferation Assay, Differentiation Assay, Control

Journal: Scientific Reports

Article Title: Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

doi: 10.1038/s41598-017-12880-9

Figure Lengend Snippet: S100B is required during the macrophage M2 phase for efficient regeneration. ( a ) Injured TA muscles were injected with IgG or anti-S100B antibody at d4 p.i. Treated muscle were excised at d7 or d14 p.i. ( b ) Histology of muscle tissue (upper panel) and counts of interstitial cells and centrally nucleated myofibers (lower panel). ( c ) PAX7 + , MyoD + , myogenin + , MAC3 + and Ki67 + cell counts (also see Fig. ). ( d ) Western blots of the indicated proteins in homogenates of IgG- and anti-S100B-treated muscles. Immunoblots of GAPDH and α-actinin are included as loading controls. ( e ) Myofiber size distribution at d14 p.i. of uninjured muscles and IgG- and anti-S100B-treated injured muscles. ( f,g ) Macrophages isolated from IgG- and anti-S100B-treated injured muscles and analyzed by real-time PCR. Full-length blots are presented in Supplementary Fig. “Fig. 3”. Results are means ± SEM (n = 6). ** p < 0.01, *** p < 0.001 vs. control. The scale bar in ( b , c and f ) represents 50 μm.

Article Snippet: At d1 or d4 p.i., TA muscles were injected with either a neutralizing rabbit polyclonal anti-S100B antibody (Abcam No. ab41548) or rabbit non-immune IgG (Sigma) (100 ng/muscle).

Techniques: Muscles, Injection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

doi: 10.1038/s41598-017-12880-9

Figure Lengend Snippet: S100B’s ability to promote regeneration of acutely injured skeletal muscles requires RAGE at early, but not mid-late regeneration phase. ( a ) Injured Ager −/− TA muscles were injected with IgG or anti-S100B antibody at d4 p.i. and excised at d7 p.i. ( b ) Counts of interstitial cells and centrally nucleated myofibers (also see Fig. ). ( c ) PAX7 + , MyoD + , myogenin + , MAC3 + and Ki67 + cell counts (also see Fig. ). ( d ) Western blots of the indicated proteins in homogenates of IgG- and anti-S100B-treated Ager –/– muscles. Immunoblots of GAPDH and α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig. “Fig. 4”. ( e , f ) Macrophages isolated at d7 p.i. from injured Ager −/− muscles and analyzed by real-time PCR to measure the indicated macrophage markers ( e ) and Tgfb ( f ). Results are means ± SEM (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Article Snippet: At d1 or d4 p.i., TA muscles were injected with either a neutralizing rabbit polyclonal anti-S100B antibody (Abcam No. ab41548) or rabbit non-immune IgG (Sigma) (100 ng/muscle).

Techniques: Muscles, Injection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

doi: 10.1038/s41598-017-12880-9

Figure Lengend Snippet: Late blockade of S100B results in altered bFGF/FGFR1 signaling. ( a , b ) Injured Ager −/− TA muscles were injected with IgG or anti-S100B antibody at d1 ( a ) or d4 ( b ) p.i. and excised at d3 or d7, respectively. Muscle homogenates were subjected to western blotting for detection of total and phosphorylated (pho-Tyr)-FGFR1. # Denotes experiment number. Full-length blots are presented in Supplementary Fig. “Fig. 5”. ( c ) Conditions were as in ( b ) except that mice were treated with vehicle or SU5402 at d3, d4, d5 and d6 p.i. Muscles were excised at d7 p.i. and analyzed by histology for counts of interstitial cells and centrally nucleated myofibers. ( d ) PAX7 + , MyoD + , myogenin + , MAC3 + and Ki67 + cell counts (also see Fig. ). ( e ) Flow cytometry analysis of CD163 and iNOS on CD11b + -F4/80 + cells isolated at d4 p.i. from Ager –/– muscles. Numbers above the gates indicate the frequency of positive cells. ( f ) Flow cytometry analysis of FGFR1 on CD11b + -F4/80 + -CD163 + and CD11b + -F4/80 + -iNOS + cells isolated at d4 p.i. from Ager −/− muscles. Numbers above the gates indicate the frequency of positive cells. ( g ) Macrophages isolated at d4 p.i. from injured Ager −/− muscles and analyzed by proximity ligation assay for detection of S100B-bFGF-FGFR1 complexes. Results are means ± SEM (n = 6). ** p < 0.01, *** p < 0.001 vs. control. The scale bar represents 50 µm in ( c ) and 100 µm in ( g ).

Article Snippet: At d1 or d4 p.i., TA muscles were injected with either a neutralizing rabbit polyclonal anti-S100B antibody (Abcam No. ab41548) or rabbit non-immune IgG (Sigma) (100 ng/muscle).

Techniques: Muscles, Injection, Western Blot, Flow Cytometry, Isolation, Proximity Ligation Assay, Control

Journal: Scientific Reports

Article Title: Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

doi: 10.1038/s41598-017-12880-9

Figure Lengend Snippet: Persistence of S100B at damage sites following acute muscle injury prolongs the M1 macrophage (inflammatory) phase and dampens muscle regeneration. ( a ) Injured wild-type or Ager −/− TA muscles were injected with vehicle or S100B at d1, d3 and d5 p.i., excised at d7 p.i. and analyzed as detailed in b-f for wild-type TA and in g for Ager −/− TA. ( b ) Histology and counts of interstitial cell and centrally nucleated myofiber numbers. ( c ) Muscle homogenates were subjected to western blotting for detection of the indicated proteins. Immunoblots of GAPDH and α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig. “Fig. 6”. ( d ) Counts of cell types based on immunohistochemistry for the indicated antigens. ( e ) Macrophages were isolated from muscles and subjected to real-time PCR for determination of levels of the indicated genes. ( f ) Muscles were analyzed for MAC3 or collagen IV expression by immunohistochemistry and western blotting. Immunoblots of α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig. “Fig. 6”. ( g ) Injured Ager −/− TA muscles were injected with S100B as described in ( a ) and analyzed as described in ( b ). Results are means ± SEM (n = 6). ** p < 0.01, *** p < 0.001 vs. control. The scale bar represents 50 µm in ( b , f and g ).

Article Snippet: At d1 or d4 p.i., TA muscles were injected with either a neutralizing rabbit polyclonal anti-S100B antibody (Abcam No. ab41548) or rabbit non-immune IgG (Sigma) (100 ng/muscle).

Techniques: Muscles, Injection, Western Blot, Immunohistochemistry, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Scientific Reports

Article Title: Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

doi: 10.1038/s41598-017-12880-9

Figure Lengend Snippet: High levels of released S100B in mdx muscles prolong the M1 macrophage inflammatory phase and dampen muscle regeneration. ( a , b ) Uninjured, untreated TA muscles of 4-wk-old wild-type and mdx mice were excised and incubated in PBS for 2 h at 4 °C. Conditioned media were collected, TCA precipitated and subjected to western blotting for detection of released S100B. M denotes purified S100B (10 ng). # Denotes experiment number. Immunoblots of GAPDH and α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig. “Fig. 7”. ( c ) Conditions were as in ( a , b ) excepting that muscles were fixed and paraffin-included. Cross-sections were subjected to immunohistochemistry (left panels) for detection of S100B. Mdx TA muscles processed as above were subjected to double immunofluorescence for detection of S100B (green) and either MAC3 (red) or myogenin (red) (right panels). ( d – i ) Four-wk-old mdx mice were i.p. injected with either IgG or anti-S100B antibody (1 µg/mouse) for 3 days every other day and sacrificed two days after the last injection ( d ). TA and QF muscles were subjected to histology for counts of normal, regenerating and regenerated myofibers ( e ), QF muscles were analyzed for myofiber size distribution ( f ), TA muscle homogenates were subjected to western blotting for detection of the indicated proteins ( g ), TA and QF muscles were subjected to either MAC3 immunohistochemistry for measurement of MAC3 + areas ( h ) or to IgG staining for measurement of necrotic areas ( i ). Immunoblots of GAPDH and α-actinin are included as loading controls. Full-length blots are presented in Supplementary Fig. “Fig. 7”. Results are means ± SEM (n = 6) ( e , h , i ). # Denotes experiment number ( g ). ** p < 0.01, *** p < 0.001 vs. control. The scale bar represents 50 µm in ( c,e,h,i ).

Article Snippet: At d1 or d4 p.i., TA muscles were injected with either a neutralizing rabbit polyclonal anti-S100B antibody (Abcam No. ab41548) or rabbit non-immune IgG (Sigma) (100 ng/muscle).

Techniques: Muscles, Incubation, Western Blot, Purification, Immunohistochemistry, Immunofluorescence, Injection, Staining, Control

Journal: Scientific Reports

Article Title: Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy

doi: 10.1038/s41598-017-12880-9

Figure Lengend Snippet: Schematic of the proposed role of S100B in muscle regeneration following acute injury and in mdx muscles. ( a ) Top panel. S100B, released from damaged myofibers and infiltrating macrophages, attracts macrophages to damage sites, promotes M1/M2 macrophage switch and stimulates myoblast proliferation RAGE-dependently during the first 3–4 days p.i. Depending on local conditions (e.g., myoblast density, S100B levels and bFGF availability) S100B can stimulate myoblast proliferation and M1/M2 macrophage switch either by engaging RAGE or by enhancing bFGF-FGFR1 signaling in this time interval and thereafter. Effects of S100B on attraction of macrophages to damage sites are strictly RAGE-dependent. Middle panel. Blocking S100B early after injury delays macrophage infiltration and M1/M2 macrophage switch with resultant delayed regeneration. Bottom panel. Defective clearance or excess release of S100B resulting in persistence of S100B at damage sites prolongs the M1 (proinflammatory) macrophage phase via RAGE engagement with resultant delayed regeneration. ( b ) In muscular dystrophy (DMD) levels of released S100B (from damaged myofibers and infiltrating macrophages) are high, which might contribute to a state of unrestricted inflammation and resultant defective regeneration (top panel). Indeed, blocking S100B in this condition reduces macrophage infiltration and inflammation and improves muscle regeneration (bottom panel).

Article Snippet: At d1 or d4 p.i., TA muscles were injected with either a neutralizing rabbit polyclonal anti-S100B antibody (Abcam No. ab41548) or rabbit non-immune IgG (Sigma) (100 ng/muscle).

Techniques: Muscles, Blocking Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Neuromuscular junction (NMJ) regions. NMJ regions were localized in muscle using a combination of silver staining, enzyme histochemistry, direct fluorescence, and immunostaining. Presynaptic regions were identified using silver staining histochemistry for acetylcholinesterase (AchE) enzyme activity and neurites (A) alone, and additionally localized in combination with staining for postsynaptic acetylcholine receptor (AchR) plaques which were identified by green fluorescence after staining with FITC-conjugated α-bungarotoxin (B–D) . Terminal Schwann cells (TSCs) localized in some sections by further immunostaining for S100B protein, identified by red fluorescence (D) . (A,B) , and (D) illustrate NMJ regions in regenerating muscle on day 6 after cardiotoxin injury (A,B) and day 8 after muscle-crush injury (D) . (C) Illustrates presynaptic neurites branching toward regions of AchE activity closely juxtaposed to postsynaptic AchR plaques in control muscle without injury. Nuclei are counterstained blue by 4′,6-diamidino-2-phenylindole (A,C,D) . Bars indicate ×10 µm.

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: Silver Staining, Fluorescence, Immunostaining, Activity Assay, Staining, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Combined in situ hybridization (ISH) experiments and P75 immunostaining. A combination of fluorescent ISH and immunostaining was used to localize the expression of Sema3A (green) and S100B (red) mRNAs and P75 protein (white) in the tibialis anterior muscle at different time points after cardiotoxin (CTX) and muscle-crush (CR) injury, with or without ISDN pretreatment (A) . CTX-injured muscle at day 0 (immediately following CTX injection and after 2 days of ISDN pretreatment) showing S100B expression (i, red fluorescence), Sema3A expression (ii, green fluorescence), and S100B and Sema3A mRNA signals merged with the differential interference contrast (DIC) image (iii) (B) . Images of ISH signals overlain with corresponding DIC images of the same representative fields of control untreated, uninjured muscles (i, ii) showing S100B (red) and Sema3A (green) ISH signals alone (i) or in combination with P75 immunostaining (white) (ii). (Bii) shows two probable terminal Schwann cells (TSCs) identified at rounded S100B-expressing regions surrounded by P75 protein (arrows). Bars indicate 10 µm (A,Bi) or 5 µm (Bii) .

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: In Situ Hybridization, Immunostaining, Expressing, Injection, Fluorescence, Control, Muscles

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Colocalization of expression. Representative in situ hybridization (ISH) images merged with differential interference contrast (DIC) of the same field, showing Sema3A (green) and S100B (red) mRNA expression after cardiotoxin injury (day 6 (D6)) (i,ii) and after muscle-crush injury (D8, iii,iv, and D10, v,vi ) without pretreatment (i,iii,v) and with 2 days of ISDN pretreatment (ii,iv,vi) . Areas of Sema3A (green) and S100B (red) mRNA expression colocalized with P75 protein (white fluorescence) in terminal Schwann cells are indicated by white circles. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (blue) in all fields. Bars indicate 10 µm.

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: Expressing, In Situ Hybridization, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Analysis of in situ hybridization (ISH) signals and areas of colocalization using Celleste software. After imaging and software calibrations for brightness and magnification, fluorescent regions in each image field were viewed. The program automatically detected, color-outlined, coded, and labeled each fluorescent site with a unique alpha-numeric identifier, before counting sites and measuring each for intensity and maximum diameter. (A,B) show two representative highly magnified fields of muscle to illustrate the labeling and counting feature of the software applied to a single fluorochrome, both at day 0 after muscle-crush injury. Fields display numerous sites outlined for red fluorescence (A) , detecting S100B (outlined by red lines), or green fluorescence (B) , detecting Sema3A (outlined by green lines), with alpha-numeric labels. Fluorescent sites varied from tiny to large. Each irregularly shaped region of fluorescence is counted as a single expression site, according to specifications of the RNAScope v2 multiplex technology. The intensity (areal density) of each region was measured (not including non-fluorescent pixels inside an outline). The maximum diameter of each fluorescent region was measured in pixels, and data for each site in every imaged field (10 per section) were recorded and compiled in a spreadsheet. (C) Shows three regions of blue fluorescence (a test area of prominent 4′,6-diamidino-2-phenylindole-labeled nuclei) that are outlined in red (one is indicated by the white arrow). Overlapping areas of green fluorescence were identified using the “smart segmentation” tool. (D) Shows the application of the Celleste “colocalization” feature used to identify and measure the spatial overlap (area) between two (or more) different fluorescent color labels. In this example, the colocalization feature identified regions of overlap between blue and green in panel (C) and indicates those as the white regions shown in (D) . Regions of colocalization were subsequently counted by the software. (E) Shows a small highly magnified area from a field imaged for ISH signals in a representative muscle section showing three sites that were outlined (dashed red lines) for red fluorescence. One of those sites (white arrow) overlaps and is surrounded by green fluorescence from a second probe (dashed green outline). White circles in (C,E) indicate regions used to measure the background intensity. In each field, background intensity was subtracted from intensity measurements of fluorescence. Bars indicate 1.8 µm (A,B,E) or 3 µm (C,D) .

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: In Situ Hybridization, Software, Imaging, Labeling, Fluorescence, Expressing, RNAscope, Multiplex Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Expression analysis of S100B and Sema3A transcripts and P75 protein in regenerating muscle. Muscle-crush (CR)- and cardiotoxin (CTX)-injured muscles in mice without treatment (control) or after 2 days of pretreatment with isosorbide dinitrate (ISDN) from 0 to 10 days after injury (Day). Celleste software measured the expression-site number, the intensity of fluorescence (areal density, arbitrary grayscale units), and the diameter (pixels) against calibration standards. Sample size ( n ) for each entry appears in brackets; “-” indicates missing data.

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: Expressing, Muscles, Control, Software, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Expression at day 0 after injury. Expression sites of S100B and Sema3A transcripts and P75 protein were identified using fluorescent in situ hybridization and immunostaining, respectively, in muscle-crush (CR)- and cardiotoxin-injured muscles from mice either treated with ISDN for 2 days before injury (+ISDN) or controls without treatment (no ISDN). In this staining experiment, n = 2 per group. The number of fluorescent expression sites was identified and counted by Celleste software, which also identified the number of sites with a colocalized expression of S100, Sema3A, and P75 (indicated as S + S + P75) (A) . At day 0, the number of Sema3A expression sites was significantly lower after CR injury in ISDN-treated mice than in the control CR group ( p = 0.038, t -test, n = 4) (B) . The number of sites expressing S100B mRNA was significantly correlated with the number of Sema3A expression sites (linear regression R 2 = 0.69, p < 0.001) (C) . The number of Sema3A expression sites was also correlated with the number of sites expressing P75 protein (linear regression R 2 = 0.39, p < 0.012). * p < 0.05.

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: Expressing, In Situ Hybridization, Immunostaining, Muscles, Staining, Software, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Principal component analysis (PCA) correlation circle of the overall dataset. Two factors or principal components (F1, F2) form the x and y axes of the PCA correlation circle, respectively. A third factor (F3) is on the z -axis, which extends upward from the x-y plane. The PCA dataset included variables from analyses of in situ hybridization (ISH), ISH with immunostaining (S100B and Sema3A transcripts and P75 protein), fiber diameter, and Western blotting (WB) assessments of S100B, Sema3A, and γAchR proteins from n = 26 mice including control and ISDN-pretreated groups after muscle-crush and cardiotoxin injury. The direction and length of the vector for each variable represent the respective loading coefficients of that variable with the three factors, F1, F2, and F3, as orthogonal axes. The endpoint position of each vector on the x-y grid represents the loading (or correlation) score of data collected for that variable (here, including all injury and treatment groups) on each of the F1 and F2 in the PCA plot. The factor to which a particular variable loads most strongly is indicated as a black line (to F1), a gray line (to F2), and gray dashes (to F3). Vectors are labeled for each variable as follows: WB for Western blotting data; BV for blood vessel density; and S + S + P75 for the number of overlapping expression sites for all three, Sema3A and S100B mRNAs and P75 protein. Data for fiber diameter, blood vessel number, and Western blotting were reported previously . provides the loading coefficients for each variable in this PCA with each of the three factors.

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: In Situ Hybridization, Immunostaining, Western Blot, Control, Plasmid Preparation, Labeling, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Loading correlations of variables onto 3 factors identified by principal component analysis of the full dataset of muscles ( n = 26) regenerating from injury with or without pretreatment by ISDN before injury. WB indicates protein assays by Western blotting, BV = blood vessel, “S + S + P75” indicates the number of regions in which P75 protein (by immunostaining) was colocalized with both S100B and Sema3A transcripts (by in situ hybridization, ISH). # indicates the number of expression sites by ISH. Loading coefficients greater than 0.5 (in bold type) were considered strong correlations.

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: Muscles, Western Blot, Immunostaining, In Situ Hybridization, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Principal component analysis (PCA) correlation circles of control and ISDN-treated groups of regenerating muscle. Factors F1 and F2 form the x and y axes of each PCA correlation circle, respectively. A third factor (F3) is on the z -axis, which extends upward from the x-y plane. PCA datasets included variables from analyses of in situ hybridization (ISH), ISH with immunostaining (S100B and Sema3A transcripts and P75 protein), fiber diameter, and Western blotting (WB) assessments of S100B, Sema3A and γAchR proteins from both muscle-crush (CR)- and cardiotoxin (CTX)-injured groups. The direction and length of the vector for each variable represent the respective loading coefficients of that variable with the three factors, F1, F2, and F3, as orthogonal axes. The endpoint position of each vector on the x-y grid represents the loading (or correlation) score of data collected for that variable (here, including all injury and treatment groups) on each of the F1 and F2 in the PCA plot. The factor to which a particular variable loads most strongly is indicated as a black line (to F1), a gray line (to F2), and gray dashes (to F3). (A) Correlation circle for untreated control muscles regenerating from CR and CTX injury ( n = 12). (B) Correlation circle for muscles regenerating from CR and CTX injury after 2 days of pretreatment with ISDN ( n = 14). Vectors are labeled for each variable as follows: WB for Western blotting data; BV for blood vessel density; and S + S + P75 for the number of overlapping expression sites for all three, Sema3A and S100B mRNAs and P75 protein. Data for fiber diameter, blood vessel number, and Western blotting were reported previously . and provide the loading coefficients of each variable in the correlation circles shown in (A,B) , respectively.

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: Control, In Situ Hybridization, Immunostaining, Western Blot, Plasmid Preparation, Muscles, Labeling, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Loading correlations of variables onto 3 factors identified by principal component analysis of the dataset of untreated muscles regenerating from injury. WB indicates data originated from Western blotting assays, BV = blood vessel; # indicates the number of expression sites (by in situ hybridization, ISH); “S + S + P75” indicates the number of regions in which P75 protein (by immunostaining) was colocalized with S100B and Sema3A transcripts (by ISH). Loading coefficients greater than 0.5 (in bold type) were considered strong correlations.

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: Muscles, Western Blot, Expressing, In Situ Hybridization, Immunostaining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Preliminary Study of S100B and Sema3A Expression Patterns in Regenerating Muscle Implicates P75-Expressing Terminal Schwann Cells and Muscle Satellite Cells in Neuromuscular Junction Restoration

doi: 10.3389/fcell.2022.874756

Figure Lengend Snippet: Loading correlations of variables onto 3 factors identified by principal component analysis of the dataset from muscles regenerating after pretreatment with ISDN before muscle-crush or cardiotoxin injury. WB indicates protein assays by Western blotting, BV = blood vessel; # indicates the number of expression sites (by in situ hybridization, ISH); “S + S + P75” indicates the number of regions in which P75 protein (by immunostaining) was colocalized with S100B and Sema3A transcripts (by ISH); intensity and diameter refer to measurements of fluorescent regions of S100B or Sema3A mRNA expression (by ISH). Loading coefficients greater than 0.5 (in bold type) were considered strong correlations.

Article Snippet: For staining for localized S100B protein on a subset of the same slides, rabbit polyclonal anti-S100B antibody (orb228251, Biorbyt, 1:3,000), blocking, and goat anti-rabbit IgG DyLight650 (ab96902, Abcam, 1:200) were used to identify TSCs.

Techniques: Muscles, Western Blot, Expressing, In Situ Hybridization, Immunostaining