Journal: Development (Cambridge, England)

Article Title: Analysis of cranial tenocyte heterogeneity reveals a role for Wnt signaling in tendon attachments

doi: 10.1242/dev.205047

Figure Lengend Snippet: Subsets of tenocytes are marked by active Wnt signaling during development. (A,F,K) Ventral views of cranial tendons at 48 hpf (A), 53 hpf (F) and 72 hpf (K) in Tg(scxa:mCherry; 7xTCF:GFP) zebrafish embryos immunostained for anti-mCherry (tenocytes, magenta), anti-GFP (Wnt responsive cells, green) and anti-MHC (muscle fibers, blue). (P) Ventral view of is HCR-stained Tg(7xTCF:GFP) 72 hpf embryo showing expression of scxa in tenocytes (magenta), sox9a in cartilage (blue) and co-immunostained with anti-GFP (Wnt responsive, green). (B-D,G-I,L-N,Q-S) Magnified views of region marked by white dotted lines in A,F,K,P, respectively, showing immunofluorescent signal of mCherry and GFP and is HCR expression of scxa , sox9a and IF staining of GFP. (E,J,O,T) Monochrome panel showing tenocytes co-expressing mCherry and GFP, and scxa and GFP. (U-W) Magnified view of region marked by red dotted lines in P showing is HCR expression of scxa , sox9a and IF staining of GFP. (X) Monochrome image showing tenocytes co-expressing scxa and GFP. Scale bars: 20 μm (A,F,K,Q,U); 30 μm (P); 15 μm (G); 10 μm (B,L).

Article Snippet: Embryos were stained using mouse monoclonal anti-Myosin heavy chain (MHC) antibody (Developmental Studies Hybridoma Bank, A4.1025; RRID:AB_528356) at 1:200 dilution, rat monoclonal anti-mCherry antibody (Invitrogen, M11217 ; RRID:AB_2536611) at 1:500 dilution, rabbit polyclonal anti p-Myosin light chain (p-MLC) antibody (Cell Signaling Technology, 3671; RRID:AB_330248) at 1:500 dilution, rabbit anti-Thrombospondin4b antibody (Thbs4b) (GeneTex, GTX125869; RRID:AB_2885605) at 1:1000 dilution.

Techniques: Staining, Expressing

Journal: Development (Cambridge, England)

Article Title: Analysis of cranial tenocyte heterogeneity reveals a role for Wnt signaling in tendon attachments

doi: 10.1242/dev.205047

Figure Lengend Snippet: Genetic perturbation of Wnt signaling disrupts muscle attachments and tendon pattern. (A-R) Ventral views of cranial tendons at 48 hpf and 60 hpf Tg(scxa:mCherry) , Tg(scxa:mCherry; hsp70l:dkk1b-GFP) and Tg(scxa:mCherry; hsp70l:dnTCF-GFP) embryos that were heat shocked and immunostained for anti-mCherry (tenocytes), anti-GFP (Wnt responsive cells) and anti-MHC (muscle fibers). Images show muscle attachments in untreated (A,J) and heat-shocked controls (B,K), Tg(scxa:mCherry) and heat-shocked transgenics with perturbed Wnt signaling (D-I, M-R). (C,L) Histograms show distribution of normal, mild and severe muscle attachment defects between control and heat-shocked Tg(scxa:mCherry; hsp70l:dkk1b-GFP) embryos. P -values were calculated using chi-square test of independence. *** P <0.001 (48 hpf, n ≈79; 60 hpf, n ≈76). Scale bars: 30 μm.

Article Snippet: Embryos were stained using mouse monoclonal anti-Myosin heavy chain (MHC) antibody (Developmental Studies Hybridoma Bank, A4.1025; RRID:AB_528356) at 1:200 dilution, rat monoclonal anti-mCherry antibody (Invitrogen, M11217 ; RRID:AB_2536611) at 1:500 dilution, rabbit polyclonal anti p-Myosin light chain (p-MLC) antibody (Cell Signaling Technology, 3671; RRID:AB_330248) at 1:500 dilution, rabbit anti-Thrombospondin4b antibody (Thbs4b) (GeneTex, GTX125869; RRID:AB_2885605) at 1:1000 dilution.

Techniques: Control

Journal: Experimental gerontology

Article Title: Sustaining neuromuscular activation after total knee arthroplasty preserves skeletal muscle fiber size, contractility, and innervation in older adults

doi: 10.1016/j.exger.2025.112831

Figure Lengend Snippet: Effect of NMES on the response of muscle fiber size to total knee arthroplasty (TKA). Representative immunohistochemistry images for pre-surgery and 5-wk post-surgery Control ( A and B , respectively) and NMES ( C and D , respectively) groups. Tissue sections treated with antibodies recognizing myosin heavy chain (MHC) I (green), MHC IIA (red), and MHC IIX (blue) isoforms are shown. As fibers expressing only MHC IIX are rare, the large majority of MHC IIX expression occurs in MHC IIAX hybrid fibers (purple). Scale bar = 50 um. Skeletal muscle fiber cross-sectional area (CSA) calculated from minimal Feret diameter ( E ) for all fiber types pooled together (All Fibers) and MHC I, IIA, and IIAX fibers are shown at pre-surgery (open bar) and post-surgery (filled bar) for patients in control (black) and NMES (blue) groups. Analyses were limited to fibers expressing MHC I, IIA, and IIAX isoforms because other MHC isotype fibers (MHC I/IIA, IIX, and I/IIA/IIX) were too few to permit analyses. Bars represent mean and SE from parameter estimates from the linear mixed model analysis for n = 10 patients for each time point derived, except for n = 9 patients for MHC IIAX post-surgery because one patient did not have any MHC IIAX fibers at one time point. Data points represent individual volunteer average values for single fibers clustered within each volunteer for baseline and 5-week post-surgery evaluations. Please note that these individual average data are presented for data transparency purposes only and are not utilized in the statistical analytical model. Bar graphs represent least squared means and standard error values derived from the mixed model analysis. Deviation of individual values from bars graph data are due to the fact model-derived values reflect adjustment for the effects of sex, as well as other factors. In addition, many of the measures were log transformed for analysis, and as such the least squared means and standard errors plotted were back transformed into original units. Thus, these values can be somewhat offset from those of the raw data. P values indicate time (T) and group X time (GxT) effects (atop brackets) or within group comparisons over time (over lines). * P < 0.05, *** P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Briefly, primary antibodies raised against the three main skeletal muscle MHC isoforms (MHC I, IIA, IIX; BA-D5, SC-71, and 6H-1 from Developmental Studies Hybridoma Bank, Iowa City, IA) are used to identify all 6 fiber isotypes (MHC I, I/IIA, IIA, IIA/X, IIX, I/IIA/IIX).

Techniques: Immunohistochemistry, Control, Expressing, Derivative Assay, Transformation Assay

Journal: Physiological Reports

Article Title: Macrophages modulate skeletal muscle wasting and recovery in acute lung injury in mice

doi: 10.14814/phy2.70052

Figure Lengend Snippet: Intramuscular (IM) macrophage depletion with clodronate liposomes on was performed starting on ALI day 6 with analysis on ALI day 10 (a) ( n = 9). Contralateral TA muscle was treated with vehicle control liposomes. (b) Tibialis Anterior (TA) wet weight ( n = 9). For morphometric analyses, the tibialis anterior muscle cryosections were stained for laminin‐γ1 and cross‐sectional area (CSA) was determined. The data were then expressed as (c) mean fiber CSA or as (d) distribution of CSA of the total number of myofibers analyzed. (e) The tibialis anterior muscle cryosections were also stained for MHC isoforms and expressed as mean fiber CSA for each subtype: Types IIX, IIA, IIB, IIX/A, and IIX/B. (f) Representative tibialis anterior muscle MHC stain with types IIX (black), IIA (green), IIB (red), IIX/A (intermediate green), and IIX/B (intermediate red). Values are expressed as mean ± SD. Wilcoxon signed‐rank test (paired nonparametric t test) was performed (b, c). For comparison of the distribution of fibers between two groups, a Poisson regression model was fitted in R (version 4.1.1 stats package) to predict the count of fibers with a size greater than 600 μm 2 , including the individual animal as a covariate and total number of fibers counted as an offset (d). Mann Whitney test (unpaired nonparametric t test) was performed between treatments for each fiber type (e). Figure 4a created with BioRender.com .

Article Snippet: To process samples for immunofluorescence of myosin heavy chain isoforms (Bonilla et al., ), sections were rinsed in PBST and blocked with 10% goat serum in PBS for 1 h. The mouse primary antibodies used for MHC isoforms, developed by Dr. Stefano Schiaffino (Schiaffino et al., ) and purchased through the University of Iowa Developmental Studies Hybridoma Bank, include BA‐F8 (MHC‐I, 1:50), SC‐71 (MHC‐IIa, 1:500), and BF‐F3, (MHC‐IIb, 1:100).

Techniques: Liposomes, Control, Staining, Comparison, MANN-WHITNEY

Journal: Science Advances

Article Title: Lmod2 is necessary for effective skeletal muscle contraction

doi: 10.1126/sciadv.adk1890

Figure Lengend Snippet: Lmod2 WT + cGFP and KO + cGFP-Lmod2 skeletal muscle cross sections were stained with antibodies against different MHC isoforms. Representative images of EDL ( A ) or soleus ( B ) MHC fiber type staining. Top: MHC type I (blue), MHC type IIB (green), and laminin (red). Middle: MHC type IIA (green) and laminin (red). Bottom: MHC type IIX (blue) and laminin (red). Quantification of the number of MHC type I–, IIA–, IIB–, or IIX–positive fibers in EDL ( C ) or soleus ( E ) cross-sections shown as percent of total fibers. Minimum Feret diameter of MHC type I–, IIA–, IIB–, or IIX–positive fibers in EDL ( D ) or soleus ( F ) cross sections. Scale bars, 100 μm. All values are means ± SD; P < 0.05 was considered significant, unpaired t test; * P < 0.05, ** P < 0.01; n = 4 mice per genotype.

Article Snippet: The following primary antibodies were used: MHC I [3.5 mg/ml; BA-F8 IgG2b, Developmental Studies Hybridoma Bank (DSHB)], MHC IIB (9 mg/ml; BF-F3 IgM, DSHB), MHC IIA (3.5 mg/ml; SC-71 IgG1, DSHB), MHC IIX exclusion (3.5 mg/ml; BF-35 IgG1, DSHB), pan-myosin (9 mg/ml; A4-1025 IgG2a, DSHB), and rabbit polyclonal laminin (1:400; L9393, Sigma-Aldrich).

Techniques: Staining