Immunostaining:Article Title: Notch signaling in osteoblast progenitor cells is required for BMP-induced bone formation.
Article Snippet: Notch signaling is active during bone formation and prior studies have shown that it is required for both robust intramembranous and endochondral bone regeneration.. Particularly, the systemic blockade of Notch signaling has been shown to inhibit BMP-induced bone formation in a murine calvarial defect model.. In this study, we genetically disrupted the expression of both the dominant Notch receptor, Jagged-1, and the essential Notch signaling transcription factor Rbpj in osteoblast progenitors during calvarial bone healing.
Immunofluorescence:Article Title: Oligodendroglia Generate Vascular Mural Cells and Neurons in the Adult Mouse Brain.
Article Snippet: .. For immunofluorescence staining, the tissues were incubated overnight at 4°C with primary antibodies targeting specific proteins: cluster of differentiation 13 (CD13, #GTX75927, Genetex, Irvine, CA, U.S.A.), Sox10 (#AF2698, Beyotime, Shanghai, China), PDGFRβ (#AF1042, R&D Systems, Minneapolis, MN, U.S.A.; #14-1402-82, Thermo Fisher, Waltham, MA, U.S.A.), smooth muscle myosin heavy chain 11 (MYH11, #ab224804, Abcam, Cambridge, U.K.), NG2 (#ab5320, Millipore, Bedford, MA, U.S.A.; #ab129051, Abcam), aquaporin-4 (AQP4, #59678, CST, Danvers, MA, U.S.A.), PDGFRα (#558774, BD Biosciences, Franklin Lakes, NJ, U.S.A.), Ki67 (#12202, CST; #ab15580, Abcam), neuronal nuclei (NeuN, #266004, Synaptic Systems, Göttingen, Germany), microtubule-associated protein 2 (MAP2, #8707, CST), RFP (#600-401-379, Rockland, Limerick, PA, U.S.A.), gamma-aminobutyric acid (GABA, #A2052, Millipore), Reelin (#ab312310, Abcam), the proto-oncogene c-Fos (#2250, CST), doublecortin (DCX, #4604, CST), vesicular glutamate transporter 2 (VGLUT2, #MAB5504, Millipore), adenomatous polyposis coli (clone CC1, #GTX16794, Genetex), activating transcription factor 3 (ATF-3, #HPA001562, Millipore), Tau-1 (#MAB3420, Millipore), heat-shock protein 70 (Hsp70, #ab181606, Abcam), neurofilament (NF-M, #OB-MMS051-02, Oasis Biofarm, Zhejiang, China), γ-histone 2AX (γ-H2AX, #C2035S, Beyotime), cluster of differentiation 31 (#550274, BD Biosciences, Franklin Lakes, NJ, U.S.A.), glial fibrillary acidic protein (GFAP, #D262817-0025, Sangon Biotech, Shanghai, China), and Sox9 (#OB-PRB049-01, Oasis Biofarm). .. After 3 washes with phosphate-buffered saline, immunofluorescence secondary antibodies (#705-095-147, #112-095-003, #111-095-003, #115-095-003, and #706-095- 148, Jackson ImmunoResearch, West Grove, PA, U.S.A.) were applied at room temperature for 1 h. Finally, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole and the brain sections were imaged using a fluorescence microscope (Olympus, Tokyo, Japan, BX41).
Staining:Article Title: Oligodendroglia Generate Vascular Mural Cells and Neurons in the Adult Mouse Brain.
Article Snippet: .. For immunofluorescence staining, the tissues were incubated overnight at 4°C with primary antibodies targeting specific proteins: cluster of differentiation 13 (CD13, #GTX75927, Genetex, Irvine, CA, U.S.A.), Sox10 (#AF2698, Beyotime, Shanghai, China), PDGFRβ (#AF1042, R&D Systems, Minneapolis, MN, U.S.A.; #14-1402-82, Thermo Fisher, Waltham, MA, U.S.A.), smooth muscle myosin heavy chain 11 (MYH11, #ab224804, Abcam, Cambridge, U.K.), NG2 (#ab5320, Millipore, Bedford, MA, U.S.A.; #ab129051, Abcam), aquaporin-4 (AQP4, #59678, CST, Danvers, MA, U.S.A.), PDGFRα (#558774, BD Biosciences, Franklin Lakes, NJ, U.S.A.), Ki67 (#12202, CST; #ab15580, Abcam), neuronal nuclei (NeuN, #266004, Synaptic Systems, Göttingen, Germany), microtubule-associated protein 2 (MAP2, #8707, CST), RFP (#600-401-379, Rockland, Limerick, PA, U.S.A.), gamma-aminobutyric acid (GABA, #A2052, Millipore), Reelin (#ab312310, Abcam), the proto-oncogene c-Fos (#2250, CST), doublecortin (DCX, #4604, CST), vesicular glutamate transporter 2 (VGLUT2, #MAB5504, Millipore), adenomatous polyposis coli (clone CC1, #GTX16794, Genetex), activating transcription factor 3 (ATF-3, #HPA001562, Millipore), Tau-1 (#MAB3420, Millipore), heat-shock protein 70 (Hsp70, #ab181606, Abcam), neurofilament (NF-M, #OB-MMS051-02, Oasis Biofarm, Zhejiang, China), γ-histone 2AX (γ-H2AX, #C2035S, Beyotime), cluster of differentiation 31 (#550274, BD Biosciences, Franklin Lakes, NJ, U.S.A.), glial fibrillary acidic protein (GFAP, #D262817-0025, Sangon Biotech, Shanghai, China), and Sox9 (#OB-PRB049-01, Oasis Biofarm). .. After 3 washes with phosphate-buffered saline, immunofluorescence secondary antibodies (#705-095-147, #112-095-003, #111-095-003, #115-095-003, and #706-095- 148, Jackson ImmunoResearch, West Grove, PA, U.S.A.) were applied at room temperature for 1 h. Finally, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole and the brain sections were imaged using a fluorescence microscope (Olympus, Tokyo, Japan, BX41).
Incubation:Article Title: Oligodendroglia Generate Vascular Mural Cells and Neurons in the Adult Mouse Brain.
Article Snippet: .. For immunofluorescence staining, the tissues were incubated overnight at 4°C with primary antibodies targeting specific proteins: cluster of differentiation 13 (CD13, #GTX75927, Genetex, Irvine, CA, U.S.A.), Sox10 (#AF2698, Beyotime, Shanghai, China), PDGFRβ (#AF1042, R&D Systems, Minneapolis, MN, U.S.A.; #14-1402-82, Thermo Fisher, Waltham, MA, U.S.A.), smooth muscle myosin heavy chain 11 (MYH11, #ab224804, Abcam, Cambridge, U.K.), NG2 (#ab5320, Millipore, Bedford, MA, U.S.A.; #ab129051, Abcam), aquaporin-4 (AQP4, #59678, CST, Danvers, MA, U.S.A.), PDGFRα (#558774, BD Biosciences, Franklin Lakes, NJ, U.S.A.), Ki67 (#12202, CST; #ab15580, Abcam), neuronal nuclei (NeuN, #266004, Synaptic Systems, Göttingen, Germany), microtubule-associated protein 2 (MAP2, #8707, CST), RFP (#600-401-379, Rockland, Limerick, PA, U.S.A.), gamma-aminobutyric acid (GABA, #A2052, Millipore), Reelin (#ab312310, Abcam), the proto-oncogene c-Fos (#2250, CST), doublecortin (DCX, #4604, CST), vesicular glutamate transporter 2 (VGLUT2, #MAB5504, Millipore), adenomatous polyposis coli (clone CC1, #GTX16794, Genetex), activating transcription factor 3 (ATF-3, #HPA001562, Millipore), Tau-1 (#MAB3420, Millipore), heat-shock protein 70 (Hsp70, #ab181606, Abcam), neurofilament (NF-M, #OB-MMS051-02, Oasis Biofarm, Zhejiang, China), γ-histone 2AX (γ-H2AX, #C2035S, Beyotime), cluster of differentiation 31 (#550274, BD Biosciences, Franklin Lakes, NJ, U.S.A.), glial fibrillary acidic protein (GFAP, #D262817-0025, Sangon Biotech, Shanghai, China), and Sox9 (#OB-PRB049-01, Oasis Biofarm). .. After 3 washes with phosphate-buffered saline, immunofluorescence secondary antibodies (#705-095-147, #112-095-003, #111-095-003, #115-095-003, and #706-095- 148, Jackson ImmunoResearch, West Grove, PA, U.S.A.) were applied at room temperature for 1 h. Finally, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole and the brain sections were imaged using a fluorescence microscope (Olympus, Tokyo, Japan, BX41).
Article Title: Conditional BCL-2 Expression in Fibroblasts Promotes Persistent Pulmonary Fibrosis which is Reversible by Therapeutic BCL-2 Inhibition
Article Snippet: Non-specific binding was reduced by incubation in 10% mouse serum and horse IgG serum from the M.O.M immunodetection blocking kit (#BMK-2202, Vector Laboratories, Burlingame, CA). .. Primary antibodies for BCL-2 (BioRad #MCA1550, mouse, 1:100 Hercules, CA), Bcl-2 (Cell Signaling Technology # 3498, rabbit, 1:500), PDGFRα (Cell Signaling, #3174S rabbit, 1:100), PDGFRβ (R&D Systems #AF1032 goat, 1:100, Minneapolis, MN), GFP (which identifies CFP; AvesLab #GFP-1020, chicken, 1:200), αSMA (Sigma Aldrich #2547, mouse, 1:500), p16 INK (Abcam #ab211542 rabbit, 1:100), p21 WAF (Abcam #ab188224 rabbit, 1:250), β-galactosidase (Cell Signaling #27198 rabbit, 1:200), CCSP (Seven Hills Bioreagents #WRAB-3950 rabbit, 1:200, Cincinnati, OH), pro-SPC (Millipore #AB3786 rabbit, 1:500), Krt8 (DSHB # AB531826 rat, 1:100), CD45 (Invitrogen, 17-0451-82 rat, 1:100), CD31 (Invitrogen, #14-0311-82 rat, 1:100) were incubated overnight at 4 o C followed by incubation with fluorescently tagged goat anti mouse-A647 (A21236), donkey anti-rabbit-A555 (A31572), goat anti rat-A555 (A21434), or donkey anti goat-A647 (A21447) secondary antibodies at 1:100 dilution (Invitrogen, Carlsbad, CA). .. TUNEL staining to detect apoptotic cells was completed prior to antibody staining per manufacturer’s instructions (Promega, Madison, WI).
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