Anti-MYH6 + Slow Skeletal Myosin Heavy chain 抗体 [EPR10891(2)] (ab185967)

Fluorescence multiplex immunohistochemical analysis of the human skeletal muscle (Formalin/PFA-fixed paraffin-embedded sections).

Panel A: merged staining of anti-delta Sarcoglycan (ab137101, red; Opal™690), anti-MYH2 (ab264036, cyan; Opal™520) and anti-MYH6 + Slow Skeletal Myosin Heavy chain (ab250868, gray; Opal™570) on human skeletal muscle. Panel B: anti-MYH2 stained on the fast twitch type 2A fibers. Panel C: anti-MYH6 + Slow Skeletal Myosin Heavy chain stained on skeletal muscle fiber subtypes. Panel D: anti-delta Sarcoglycan stained on membrane of skeletal muscle. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

The section was incubated in three rounds of staining: in the order of ab137101 at 1/1000 dilution (1.043 μg/ml), ab264036 at 1/1000 dilution (1.116 μg/ml) and ab250868 1/5000 dilution (0.209 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. DAPI (blue) was used as a nuclear counter stain.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using the same antibody clone in a different buffer formulation (ab250868).

Lane 1 : Anti-MYH6 + Slow Skeletal Myosin Heavy chain antibody [EPR10891(2)] (ab185967) at 1/1000 dilution
Lane 2 : Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) at 1/1000 dilution

All lanes : His tagged recombinant Human Myosin-6 protein(aa 1193-1337)

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG,(H+L), Peroxidase conjugated)

Predicted band size: 224 kDa

ab185967 could recognize both Myosin 6 and 7 protein

Lane 1 : Anti-MYH6 + Slow Skeletal Myosin Heavy chain antibody [EPR10891(2)] (ab185967) at 1/1000 dilution
Lane 2 : Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) at 1/1000 dilution (His Tag)

All lanes : His tagged recombinant Human Myosin-7 protein(aa 1191-1335)

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 224 kDa

ab185967 could recognize both Myosin 6 and 7 protein

All lanes : Anti-MYH6 + Slow Skeletal Myosin Heavy chain antibody [EPR10891(2)] (ab185967) at 1/500 dilution

Lane 1 : Human adult heart lysate
Lane 2 : Mouse heart lysate
Lane 3 : Rat heart lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti Rabbit IR680 at 1/10000 dilution

Predicted band size: 224 kDa

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab185967 overnight at 4°C. Antibody binding was detected using Goat anti Rabbit IR680 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

Immunohistochemical analysis of paraffin embedded Human skeletal muscle tissue labeling Slow Skeletal Myosin Heavy chain with ab185967 at 1/2000 dilution. Counterstained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.