Anti-Myogenin antibody [F5D]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Myogenin antibody [F5D] (AB1835)

Immunofluorescence staining of Myogenin using ab1835 in ab277612, which were differentiated for 3 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab1835 at 1 µg/mL and ab6046, rabbit polyclonal to beta Tubulin, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150088, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab1835 gave comparable results using MeOH fixation (100%, 5 min).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Myogenin antibody [F5D] (AB1835)

IHC image of Myogenin staining in a section of formalin-fixed paraffin-embedded normal Human Rhabdomyosarcoma* performed on a Leica BOND™ system using the standard Protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab1835, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Myogenin antibody [F5D] (AB1835)

ab1835 staining Myogenin in undifferentiated C2C12 cells (top panel) and 2 days differentiated C2C12 cells (bottom panel).

The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab1835 at 1 μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution overnight at 4°C. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-FoFr

Collaborator

Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Myogenin antibody [F5D] (AB1835)

Adult mouse muscle section stained with ab1835. The animals were perfused with 4% PFA. The sections were incubated in 5% normal donkey serum in 0.1% PBS- and triton X100 for 1h to permeabilise the cells and block non-specific protein-protein interactions. The sections were then incubated with the antibody (ab1835, 1μg/ml) overnight at +4°C. The secondary antibody Alexa Fluor^® 568 donkey anti-mouse IgG (H+L) (red) was used at a 1/1000 dilution for 1h.

This image is courtesy of Ruma Raha-Chowdhury, University Of Cambridge, United Kingdom

• WB

Lab

Western blot - Anti-Myogenin antibody [F5D] (AB1835)

C2C12 cells were differentiated into myotubes as previously described in PMID : 26563778.

Lanes 1-7 : Merged signal (red and green). Green - ab1835 observed at 30kDa. Red - loading control ab181602 observed at 37kDa.

This blot was produced using a 4-12% Bis-tris under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was blocked for an hour using 3% milk before ab1835 and ab181602 (Rabbit anti GAPDH loading control), were incubated overnight at 4°C at a 1 in 400 dilution and 1/20000 dilution respectively. Antibody binding was detected using Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Myogenin antibody [F5D] (ab1835)

Lane 1:

Differentiated C2C12 whole cell lysate - Day 0 control at 20 µg

Lane 2:

Differentiated C2C12 whole cell lysate - Day 1 at 20 µg

Lane 3:

Differentiated C2C12 whole cell lysate - Day 2 at 20 µg

Lane 4:

Differentiated C2C12 whole cell lysate - Day 3 at 20 µg

Lane 5:

Differentiated C2C12 whole cell lysate - Day 4 at 20 µg

Lane 6:

Differentiated C2C12 whole cell lysate - Day 5 at 20 µg

Lane 7:

Differentiated C2C12 whole cell lysate - Day 6 at 20 µg

Predicted band size: 25 kDa

Observed band size: 34 kDa

false

• IHC

CiteAb

Immunohistochemistry - Anti-Myogenin antibody [F5D] (AB1835)

Immunohistochemistry using Anti-Myogenin antibody [F5D], ab1835. Publication image from Zhang, K. et al., 2018, Nat Commun, 30510196. Legend direct from paper.

The failure of Islr-deficient myoblast differentiation is rescued by canonical Wnt signaling activation. a Western blot analysis of Axin1 protein levels in shCtrl and shIslr C2C12 cells incubated with Wnt3a or saline for 24 h after 2 d in differentiation medium. b Western blot analysis of active β-catenin protein levels in nuclear lysates extracted from shCtrl and shIslr C2C12 cells incubated with Wnt3a or saline for 24 h after 2 d in differentiation medium. c Intramuscular injection of Wnt3a at 1.5 d postinjury and Western blot analysis of Axin1 protein levels in injured TA muscles of control and Islr cKO mice at 4 d postinjury. d Intramuscular injection of Wnt3a at 1.5 d postinjury and Western blot analysis of active β-catenin protein levels in injured TA muscles of control and Islr cKO mice at 4 d postinjury. e Western blot analysis of Axin1 and GSK3β protein levels in shIslr C2C12 cells incubated with 1-AKP or DMSO after 7 d in differentiation medium. f Immunofluorescence staining for MyHC in shIslr C2C12 cells treated with 1-AKP or DMSO after 7 d in differentiation medium. N = 3 cell cultures in each group. The percentages of MyHC+ cells are shown on the right. g Immunofluorescence staining for MyoG in shCtrl and shIslr C2C12 cells treated with 1-AKP or DMSO after 3 d in differentiation medium. N = 3 cell cultures in each group. The percentages of MyoG+ cells are shown on the right. h Immunofluorescence staining for MyoG in shCtrl and shIslr C2C12 cells treated with CHIR or DMSO after 3 d in differentiation medium. N = 3 cell cultures in each group. The percentages of MyoG+ cells are shown on the right. i Immunofluorescence staining for MyoG in primary myoblasts of control and Islr cKO mice treated with CHIR or DMSO after 1 d in differentiation medium. N = 3 cell cultures in each group. The percentages of MyoG+ cells are shown on the right. Scale bars are all 50 µm. Error bars represent the means ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test

• IF

CiteAb

Immunofluorescence - Anti-Myogenin antibody [F5D] (AB1835)

Immunofluorescence using Anti-Myogenin antibody [F5D], ab1835. Publication image from Zhang, K. et al., 2018, Nat Commun, 30510196. Legend direct from paper.

The differentiation ability of satellite cells is reduced in Islr KO mice in vitro. a Immunofluorescence analysis of MyoG+ cells in isolated EDL myofibers of control and Islr cKO mice after 3 d of culture. N = 3 in each group. Approximately, 30 clusters total in each group. Scale bar = 25 µm. The percentages of MyoG+ cells in the clusters are shown on the right. b Immunofluorescence staining for MyoG in FACS-purified satellite cells from control and Islr cKO mice cultured for 4 d in proliferation medium followed by 1 d in differentiation medium. N = 3 cell cultures in each group. Scale bar = 25 µm. The percentage of MyoG+ cells is shown on the right. c Western blot analysis of MyoG protein levels in isolated satellite cells of control and Islr cKO mice cultured for 4 d in proliferation medium followed by 1 d in differentiation medium. d Immunofluorescence staining for MyHC in FACS-purified satellite cells from control and Islr cKO mice cultured for 4 d in proliferation medium followed by 2 d in differentiation medium. N = 3 cell cultures in each group. Scale bar = 50 µm. The percentages of nuclei contained in the myotubes (a MyHC+ cell with at least two nuclei) are shown on the right. e The distribution of nuclei present in a MyHC+ cell differentiated from FACS-purified satellite cells of control and Islr cKO mice. N = 3 cell cultures in each group. f Western blot analysis of MyHC protein levels in isolated satellite cells of control and Islr cKO mice at 4 d in proliferation medium or 2 d in differentiation medium. Error bars represent the means ± s.d. NS : not significant, **P < 0.01, ***P < 0.001; Student’s t test

• IF

CiteAb

Immunofluorescence - Anti-Myogenin antibody [F5D] (AB1835)

Immunofluorescence using Anti-Myogenin antibody [F5D], ab1835. Publication image from Zhang, K. et al., 2018, Nat Commun, 30510196. Legend direct from paper.

The differentiation ability of satellite cells is reduced in Islr KO mice in vitro. a Immunofluorescence analysis of MyoG+ cells in isolated EDL myofibers of control and Islr cKO mice after 3 d of culture. N = 3 in each group. Approximately, 30 clusters total in each group. Scale bar = 25 µm. The percentages of MyoG+ cells in the clusters are shown on the right. b Immunofluorescence staining for MyoG in FACS-purified satellite cells from control and Islr cKO mice cultured for 4 d in proliferation medium followed by 1 d in differentiation medium. N = 3 cell cultures in each group. Scale bar = 25 µm. The percentage of MyoG+ cells is shown on the right. c Western blot analysis of MyoG protein levels in isolated satellite cells of control and Islr cKO mice cultured for 4 d in proliferation medium followed by 1 d in differentiation medium. d Immunofluorescence staining for MyHC in FACS-purified satellite cells from control and Islr cKO mice cultured for 4 d in proliferation medium followed by 2 d in differentiation medium. N = 3 cell cultures in each group. Scale bar = 50 µm. The percentages of nuclei contained in the myotubes (a MyHC+ cell with at least two nuclei) are shown on the right. e The distribution of nuclei present in a MyHC+ cell differentiated from FACS-purified satellite cells of control and Islr cKO mice. N = 3 cell cultures in each group. f Western blot analysis of MyHC protein levels in isolated satellite cells of control and Islr cKO mice at 4 d in proliferation medium or 2 d in differentiation medium. Error bars represent the means ± s.d. NS : not significant, **P < 0.01, ***P < 0.001; Student’s t test

• IF

CiteAb

Immunofluorescence - Anti-Myogenin antibody [F5D] (AB1835)

Immunofluorescence using Anti-Myogenin antibody [F5D], ab1835. Publication image from Zhang, K. et al., 2018, Nat Commun, 30510196. Legend direct from paper.

Absence of satellite cell differentiation in regenerating TA muscles of Islr cKO mice. a Immunofluorescence analysis of eMyHC+ fibers in TA muscles of control and Islr cKO mice at 3.5 (N = 3) and 5 (N = 3) d postinjury. The percentage of area occupied by eMyHC+ fibers per field at 5 d postinjury is shown on the right. b Immunofluorescence analysis of MyoG+ cells in TA muscles of control and Islr cKO mice at 5 d post injury. N = 3 in each group. The number of MyoG+ cells per myofiber is shown on the right. c Western blot analysis of eMyHC and MyoG protein levels in control and Islr cKO mice at 3.5 d postinjury. d Western blot analysis of eMyHC and MyoG protein levels in control and Islr cKO mice at 5 d postinjury. Scale bars are all 50 µm. Error bars represent the means ± s.d. **P < 0.01, ***P < 0.001; Student’s t test

• IF

CiteAb

Immunofluorescence - Anti-Myogenin antibody [F5D] (AB1835)

Immunofluorescence using Anti-Myogenin antibody [F5D], ab1835. Publication image from Zhang, K. et al., 2018, Nat Commun, 30510196. Legend direct from paper.

The failure of Islr-deficient myoblast differentiation is rescued by canonical Wnt signaling activation. a Western blot analysis of Axin1 protein levels in shCtrl and shIslr C2C12 cells incubated with Wnt3a or saline for 24 h after 2 d in differentiation medium. b Western blot analysis of active β-catenin protein levels in nuclear lysates extracted from shCtrl and shIslr C2C12 cells incubated with Wnt3a or saline for 24 h after 2 d in differentiation medium. c Intramuscular injection of Wnt3a at 1.5 d postinjury and Western blot analysis of Axin1 protein levels in injured TA muscles of control and Islr cKO mice at 4 d postinjury. d Intramuscular injection of Wnt3a at 1.5 d postinjury and Western blot analysis of active β-catenin protein levels in injured TA muscles of control and Islr cKO mice at 4 d postinjury. e Western blot analysis of Axin1 and GSK3β protein levels in shIslr C2C12 cells incubated with 1-AKP or DMSO after 7 d in differentiation medium. f Immunofluorescence staining for MyHC in shIslr C2C12 cells treated with 1-AKP or DMSO after 7 d in differentiation medium. N = 3 cell cultures in each group. The percentages of MyHC+ cells are shown on the right. g Immunofluorescence staining for MyoG in shCtrl and shIslr C2C12 cells treated with 1-AKP or DMSO after 3 d in differentiation medium. N = 3 cell cultures in each group. The percentages of MyoG+ cells are shown on the right. h Immunofluorescence staining for MyoG in shCtrl and shIslr C2C12 cells treated with CHIR or DMSO after 3 d in differentiation medium. N = 3 cell cultures in each group. The percentages of MyoG+ cells are shown on the right. i Immunofluorescence staining for MyoG in primary myoblasts of control and Islr cKO mice treated with CHIR or DMSO after 1 d in differentiation medium. N = 3 cell cultures in each group. The percentages of MyoG+ cells are shown on the right. Scale bars are all 50 µm. Error bars represent the means ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test