Journal: Diagnostics

Article Title: A User-Friendly Approach for Routine Histopathological and Morphometric Analysis of Skeletal Muscle Using CellProfiler Software

doi: 10.3390/diagnostics12030561

Figure Lengend Snippet: MyoProfiler: detection and segmentation of myofibers using anti-laminin, α1-stained muscle cross-sections. ( A ) Workflow of the first part of MyoProfiler pipeline. ( B ) RescaleIntensity module for laminin signal. Scale bar = 100 µm. ( C ) Custom “unsharp mask” step (GaussianFilter and ImageMath 1+2) for sharpening myofiber boundaries. ( D ) Line structures and myofiber boundaries are further enhanced with 4 sequential modules (two EnhanceAndSuppressFeatures modules and two ImageMath modules). ( E ) Closing module closes the intensity gaps between pixels. ( F ) MedianFilter module reduces salt-and-pepper background noise. ( G ) Threshold module detects positive signal and produces a binary image. ( H ) Morph module closes the gaps between muscle fibers and fills small holes. ( I ) Inverted binary image (ImageMath 5). ( J ) Color map image of segmented myofibers with IdentifyPrimaryObjects module. FilterObjects module discards wrong muscle fibers (magenta arrows point to discarded fibers). ( K ) OverlayOutlined generated an output image in which both segmented (red) and discarded (yellow outlines and pointed by cyan arrows) myofibers are outlined.

Article Snippet: Primary antibodies used for this study were rabbit polyclonal antibody raised against laminin, α1 (Sigma-Aldrich, Merck KGaA, Burlington, MA, USA; Cat#: L9393, RRID:AB_477163, 1:500) and rat monoclonal antibody raised against F4/80 (Bio-Rad Laboratories, Hercules, CA, USA; Cat#: MCA497G, RRID:AB_872005, 1:300).

Techniques: Staining, Generated

Journal: Diagnostics

Article Title: A User-Friendly Approach for Routine Histopathological and Morphometric Analysis of Skeletal Muscle Using CellProfiler Software

doi: 10.3390/diagnostics12030561

Figure Lengend Snippet: Quantitative measurements automatically performed with MyoProfiler and compared with Fiji software. ( A ) Representative immunofluorescence images of tibialis anterior sections from wild-type (WT) and mdx mice. Sections were stained with anti-laminin, α1 (green) and anti-F4/80 (red) antibodies and counterstained with DAPI (blue). Scale bar = 50 µm. ( B ) Minimum Feret diameter (MFD) quantification and fiber size distribution of myofibers. ( C ) Quantification of the number of nuclei. ( D ) Quantification of centrally nucleated (CNFs) and peripherally nucleated fibers (PNF). ( E ) Quantification of the number of nuclei per CNF performed by CellProfiler. ( F ) Quantification of the percentage of macrophages. Data are expressed as mean ± SEM, and unpaired t -test was used for comparison (N = 3 for WT and N = 4 for mdx ; * = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Article Snippet: Primary antibodies used for this study were rabbit polyclonal antibody raised against laminin, α1 (Sigma-Aldrich, Merck KGaA, Burlington, MA, USA; Cat#: L9393, RRID:AB_477163, 1:500) and rat monoclonal antibody raised against F4/80 (Bio-Rad Laboratories, Hercules, CA, USA; Cat#: MCA497G, RRID:AB_872005, 1:300).

Techniques: Software, Immunofluorescence, Staining, Comparison

Journal: International Journal of Molecular Medicine

Article Title: Laminin α1 as a target for the treatment of epidural fibrosis by regulating fibrotic mechanisms

doi: 10.3892/ijmm.2022.5205

Figure Lengend Snippet: Primers for reverse transcription-quantitative PCR.

Article Snippet: The siRNA against laminin α1 (5′-TGCC ATA GAT GGC ACC AAT AAC T-3′) and the corresponding negative control siRNA (5′-GGC TCT AGA AAA GCC TAT GC-3′) were purchased from Guangzhou RiboBio, Co., Ltd. Human fibroblasts (60% confluence) were transfected with 50 nM siRNA (siRNA against laminin α1 for siRNA group, and negative control siRNA for negative control group) for 48 h at 37°C with Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) and Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) to knock down the expression of laminin α1.

Techniques: Sequencing

Journal: International Journal of Molecular Medicine

Article Title: Laminin α1 as a target for the treatment of epidural fibrosis by regulating fibrotic mechanisms

doi: 10.3892/ijmm.2022.5205

Figure Lengend Snippet: Laminin α1 expression is positively and time dependently increased in response to laminectomy. (A) Immunohistochemical staining of laminin α1 in epidural fibrosis tissues, the expression of laminin α1 increased in a time-dependent manner. Results are shown as the mean OD value. Magnification, ×200. (B) Immunohistochemical staining of α-SMA in epidural fibrosis tissues; there was no significant difference among the three groups. Results are shown as the mean OD value. Magnification, ×200. Data are presented as the mean ± standard deviation. * P<0.05. α-SMA, α smooth muscle actin; IHC, immunohistochemistry; NS, not significant; OD, optical density.

Article Snippet: The siRNA against laminin α1 (5′-TGCC ATA GAT GGC ACC AAT AAC T-3′) and the corresponding negative control siRNA (5′-GGC TCT AGA AAA GCC TAT GC-3′) were purchased from Guangzhou RiboBio, Co., Ltd. Human fibroblasts (60% confluence) were transfected with 50 nM siRNA (siRNA against laminin α1 for siRNA group, and negative control siRNA for negative control group) for 48 h at 37°C with Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) and Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) to knock down the expression of laminin α1.

Techniques: Expressing, Immunohistochemical staining, Staining, Standard Deviation, Immunohistochemistry

Journal: International Journal of Molecular Medicine

Article Title: Laminin α1 as a target for the treatment of epidural fibrosis by regulating fibrotic mechanisms

doi: 10.3892/ijmm.2022.5205

Figure Lengend Snippet: Transfection efficiency of laminin α1 siRNA determined by immunofluorescence staining and RT-qPCR. (A) Expression levels of laminin α1 were measured by immunofluorescence staining among the three groups; the fluorescence density was significantly decreased in the siRNA group. No statistical significance was observed between the control and negative control groups. Magnification, ×200. (B) Results of RT-qPCR; the mRNA expression levels of laminin α1 were downregulated in the siRNA group. GAPDH was used as the control. Data are presented as the mean ± standard deviation. * P<0.05. NS, not significant; siRNA, small interfering RNA; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: The siRNA against laminin α1 (5′-TGCC ATA GAT GGC ACC AAT AAC T-3′) and the corresponding negative control siRNA (5′-GGC TCT AGA AAA GCC TAT GC-3′) were purchased from Guangzhou RiboBio, Co., Ltd. Human fibroblasts (60% confluence) were transfected with 50 nM siRNA (siRNA against laminin α1 for siRNA group, and negative control siRNA for negative control group) for 48 h at 37°C with Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) and Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) to knock down the expression of laminin α1.

Techniques: Transfection, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Fluorescence, Control, Negative Control, Standard Deviation, Small Interfering RNA, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Laminin α1 as a target for the treatment of epidural fibrosis by regulating fibrotic mechanisms

doi: 10.3892/ijmm.2022.5205

Figure Lengend Snippet: Laminin α1 regulates the proliferation, apoptosis and migration of fibroblasts, and affects their viability. (A) Fibroblast proliferation was detected using the EdU incorporation assay. The proliferative rate was decreased after laminin α1 was knocked down. Magnification, ×200. (B) Protein expression levels of PCNA, cyclin D1, Bax, Bcl-2, MMP-2 and MMP-9 were detected by western blotting in each group. (C) Fibroblast apoptosis was detected by Annexin V-FITC/PI double labeling and the apoptotic rates are presented. (D) Fibroblast viability was detected using the Cell Counting Kit-8 assay. (E) Fibroblast migration was detected using the scratch wound assay (magnification, ×40). Data are presented as the mean ± standard deviation. * P<0.05. MMP, matrix metalloproteinase; NS, not significant; PCNA, proliferating cell nuclear antigen; siRNA, small interfering RNA.

Article Snippet: The siRNA against laminin α1 (5′-TGCC ATA GAT GGC ACC AAT AAC T-3′) and the corresponding negative control siRNA (5′-GGC TCT AGA AAA GCC TAT GC-3′) were purchased from Guangzhou RiboBio, Co., Ltd. Human fibroblasts (60% confluence) were transfected with 50 nM siRNA (siRNA against laminin α1 for siRNA group, and negative control siRNA for negative control group) for 48 h at 37°C with Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) and Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) to knock down the expression of laminin α1.

Techniques: Migration, Expressing, Western Blot, Labeling, Cell Counting, Scratch Wound Assay Assay, Standard Deviation, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: Laminin α1 as a target for the treatment of epidural fibrosis by regulating fibrotic mechanisms

doi: 10.3892/ijmm.2022.5205

Figure Lengend Snippet: Laminin α1 interferes with activation of the AKT/mTOR signaling pathway. Protein expression levels of p-AKT, AKT, mTOR and p-mTOR were detected by western blotting in each group. Data are presented as the mean ± standard deviation. * P<0.05. mTOR, mechanistic target of rapamycin; NS, not significant; p-, phosphorylated; siRNA, small interfering RNA.

Article Snippet: The siRNA against laminin α1 (5′-TGCC ATA GAT GGC ACC AAT AAC T-3′) and the corresponding negative control siRNA (5′-GGC TCT AGA AAA GCC TAT GC-3′) were purchased from Guangzhou RiboBio, Co., Ltd. Human fibroblasts (60% confluence) were transfected with 50 nM siRNA (siRNA against laminin α1 for siRNA group, and negative control siRNA for negative control group) for 48 h at 37°C with Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) and Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) to knock down the expression of laminin α1.

Techniques: Activation Assay, Expressing, Western Blot, Standard Deviation, Small Interfering RNA