ly6g gr1  (Bio-Rad)

Journal: Molecular Metabolism

Article Title: Leukocyte-type 12/15-lipoxygenase is essential for timely inflammation-resolution and effective tissue regeneration following skeletal muscle injury

doi: 10.1016/j.molmet.2025.102224

Figure Lengend Snippet: Alox15 −/− mice are deficient in intramuscular 12/15-LOX-derived lipid mediators and display chronic low-grade muscle inflammation. A: Western blot analysis of the abundance of the 12/15-LOX protein in TA muscle homogenates from WT and Alox15 −/− mice. A spleen homogenate from a WT mouse served as a positive control. B: Densitometry quantification of the abundance of the 12/15-LOX protein in the TA muscle. Protein expression was normalized to GAPDH abundance. C: Alox1 5 mRNA expression in the TA muscle as determined by real-time quantitative reverse transcription PCR (RT-qPCR). Gene expression was normalized to Gapdh . D: Heatmap of the top 30 most differentially abundant lipid mediators TA muscle homogenates between WT and Alox15 −/− mice as identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). E-G: Quantification of the intramuscular concentration (pg/mg) of major 12/15-LOX metabolites including 12-hydroxy-eicosatetraenoic acid (12-HETE) ( E ), 15-hydroxy-eicosatetraenoic acid (15-HETE) ( F ), and 14-hydroxy-docosahexaenoic acid (14-HDoHE) ( G ) in TA muscle homogenates as determined by LC-MS/MS. H: TA muscle cross-sections were stained with primary antibodies against the neutrophil marker GR1, the pan monocyte/macrophage marker CD68, or the M2 macrophage marker CD163. Scale bars are 100 μm. I–K: Quantitative analysis of intramuscular numbers of neutrophils (GR1 + cells) ( I ), monocyte/macrophages (CD68 + cells) ( J ), and M2-like macrophages (CD68 + CD163 + cells) ( K ). L-M: Quantification of percentage TA muscle fiber type ( L ) and fiber type specific myofiber cross-sectional area (CSA) ( M ) as determined by MuscleJ 1.0.2 software. Bars show the mean ± SEM of 4–5 mice per group (biological replicates) with dots representing data from each individual mouse. P- values were determined by two-tailed unpaired t-tests. ∗ p < 0.05, ∗∗ p < 0.01 for WT vs. Alox15 −/− mice.

Article Snippet: Primary antibodies used include MyHC type I [Developmental Studies Hybridoma Bank (DSHB), BA-D5c, 1:100], MyHC type IIA (DSHB, SC-71c, 1:100), MyHC type IIB (DSHB, BF–F3c, 1:100), eMHC (DSHB, F1.652s, 1:20), Ly6G (GR1) (Bio-Rad, MCA2387, 1:50), CD68 (Bio-Rad, MCA1957, 1:200), CD163 (Santa Cruz, sc-58965, 1:200), and laminin (Abcam, ab7463, 1:200).

Techniques: Derivative Assay, Western Blot, Positive Control, Expressing, Reverse Transcription, Quantitative RT-PCR, Gene Expression, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Staining, Marker, Software, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Leukocyte-type 12/15-lipoxygenase is essential for timely inflammation-resolution and effective tissue regeneration following skeletal muscle injury

doi: 10.1016/j.molmet.2025.102224

Figure Lengend Snippet: Leukocyte-type 12/15-LOX deficient mice display greater local inflammation and an imbalance of pro-inflammation vs. anti-inflammatory/pro-resolving lipid mediators following acute skeletal muscle injury. A-E: Tibialis anterior (TA) muscle mRNA expression of the pan myeloid cell marker CD11b ( Itgam ) ( A ), the pan monocyte/MФ marker F4/80 ( Adgre1 ) ( B ), the M2 MФ marker CD206 ( Mrc1 ) ( C ), the hematopoietic growth factor M-CSF ( Csf1 ) ( D ), and the hematopoietic growth factor GM-CSF ( Csf2 ) ( E ) in wild type (WT) and Alox15 −/− mice at day 0 (D0), day 3 (D3), day 5 (D5), and day 14 (D14) following myofiber injury induced by intramuscular injection of 50 μL of 1.2% barium chloride (BaCl 2 ). Expression of genes of interest was normalized to Gapdh . F: Immunofluorescence staining with primary antibodies against neutrophils (PMNs) (GR1), monocytes/MФ (CD68), and M2 MФ (CD163) in injured TA muscle of WT and Alox15 −/− mice on D3 and D5 post-injury. Cell nuclei were stained with DAPI and a primary antibody against laminin was used to identify muscle fiber boundaries. Scale bars are 100 μm. G-I : Quantification of PMNs (GR1 + cells/mm 2 ) ( G ), monocytes/MФ (CD68 + cells/mm 2 ) ( H ), and M2 MФ (CD68 + CD163 + cells/mm 2 ) ( I ) at D0, D3, and D5 post-injury in WT and Alox15 −/− mice. J-N: TA mRNA expression of major lipid mediator biosynthesis enzymes including COX-1 ( Ptgs1 ) ( J ), COX-2 ( Ptsg2 ) ( K ), 5-LOX ( Alox5 ) ( L ), 12-LOX ( Alox12 ) ( M ), and 15-LOX ( Alox15 ). O: Heatmap of the top 30 most differentially abundant lipid mediators between WT and Alox15 −/− mice as identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of TA muscle homogenates. P-Q: Quantification of major representative metabolites of the COX pathway (e.g., PGE 2 ) ( P ), 5-LOX pathway (e.g., 5-HETE) ( Q ), 12-LOX pathway (e.g., 14-HDoHE) ( R ), 15-LOX pathway (e.g., 17-HDoHE) ( S ), CYP pathway [e.g., 11(12)-EpETrE) ( T ), and downstream bioactive SPMs (e.g., RvD6) ( Q ). Bars show the mean ± SEM of 4–5 mice per group (biological replicates) with dots representing data from each individual mouse. P- values were determined by two-way ANOVA followed by Holm-Šídák post-hoc tests. ∗ p < 0.05 vs. D0 and # p < 0.05 for WT vs. Alox15 −/− mice.

Article Snippet: Primary antibodies used include MyHC type I [Developmental Studies Hybridoma Bank (DSHB), BA-D5c, 1:100], MyHC type IIA (DSHB, SC-71c, 1:100), MyHC type IIB (DSHB, BF–F3c, 1:100), eMHC (DSHB, F1.652s, 1:20), Ly6G (GR1) (Bio-Rad, MCA2387, 1:50), CD68 (Bio-Rad, MCA1957, 1:200), CD163 (Santa Cruz, sc-58965, 1:200), and laminin (Abcam, ab7463, 1:200).

Techniques: Expressing, Marker, Injection, Immunofluorescence, Staining, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy