Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 23°C
Sample
Cow Cell (Skeletal Muscle)
Specification
Skeletal Muscle
Permeabilization
No
Fixative
Heat Fix
Other product details
Incubation time
1 hour(s) and 0 minute(s) · Temperature: 23°C
Dilution
1/2000
Secondary antibody
Name
Non-Abcam antibody was used: AlexFlour 594 Goat anti-Mouse IgG (H+L) Antibody
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 594
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 594
Dilution
1/1000
Additional data
Additional Notes
Utilizing the myoglobin antibody were able to see differences in relative fluorescence between fibers. Our muscle samples were derived from the longissimus muscle of a commercial beef feedlot steer. Samples were sectioned at 5´m on frost resistant slides and heat fixed onto the slide at 40?C for 20 min. Sections were then blocked for non-specific antibody binding with 5% Horse Serum / 0.2% Triton X-100 diluted in phosphate buffered saline (PBS) for 30 min at RT. Samples were incubated in the following primary antibodies for 1 hour at RT. Primary antibodies consisted of myoglobin (Cat# ab47702) and dystrophin (Cat# PA137587 Thermo Fisher) and were diluted in blocking solution at 1:2000 and 1:500, respectively. Muscle sections were rinsed with PBS 3 times to remove any residual primary antibody solution before they were incubated in the secondary antibody solution for 30 min at RT. Secondary antibodies consisted of AlexFluor 594 Goat Anti-mouse (Cat#A11005 Invitrogen) and AlexaFluor 488 Goat Anti-rabbit (Cat# A1108 Invitrogen) diluted 1:1000 in blocking solution. Hoechst dye (Cat# 33342 Invitrogen) was included in the secondary antbody solution diluted 1:1000 to identify all nuclei. Samples were again rinsed 3 times in PBS. After a final rinse with PBS, slides were cover-slipped and photomicrographs were captured using a Nikon Eclipse TI-U inverted microscope equipped with a DS-QiMC digital camera at a 10¿ working distance magnification (Nikon Instruments Inc., Melville, NY). In the attached image the myoglobin antibody is represented in red, dystrophin antibody is the green outline of the cells, and nuclei are blue.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
DR. John Gonzalez
Verified customer
提交于 May 15 2014