To assess gap junction density, immunohistochemistry was performed on two representative slides from each heart. Slides containing the largest area of hydrogel were used in the hydrogel group, and equivalent anatomical locations were used in non-injected and saline-injected hearts. Cryosections were fixed with acetone, incubated with anti-connexin 43 antibody (EMD Millipore, Billerica, MA; 1:200 dilution) and anti-cardiac troponin antibody (Abcam, Cambridge, United Kingdom; 1:100 dilution), and then stained with Alexa Fluor 568 anti-mouse antibody (Invitrogen, Carlsbad, CA; 1:500 dilution) and Alexa Fluor 488 anti-rabbit antibody (Invitrogen, Carlsbad, CA; 1:500 dilution). Nuclei were visualized with fluorescent Hoechst 33342. Slides were imaged using a Leica DM6000B (Leica Microsystems Inc, Buffalo Grove, IL) immune-fluorescent microscope at 20x magnification and analyzed using a proprietary Leica software suite. Gap junction densities in the hydrogel groups were measured in the injection regions as well as in a region on the LV free wall adjacent to the injection. A representative area within the LV free wall was selected for analysis in all other groups. Gap junction density was calculated as:
Alexa fluor 568 anti mouse antibody
Manufactured by Thermo Fisher Scientific
Alexa Fluor 568 anti-mouse antibody is a fluorescent-labeled secondary antibody used to detect and visualize primary mouse antibodies in various immunoassays and imaging applications. It is designed to provide a bright, stable fluorescent signal for the detection of target proteins or other biomolecules.
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Lab products found in correlation
Dm6000b, by Leica (1 mentions)
Alexa fluor 488 anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
A2052, by Merck Group (1 mentions)
Ab9474, by Abcam (1 mentions)
2 protocols using alexa fluor 568 anti mouse antibody
1
Quantifying Gap Junction Density in Cardiac Tissue
2
Immunofluorescence Analysis of GABA and AR in LNCaP Cells
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LNCaP cells were transfected as mentioned above, cells were collected every 24 h for 96 h. Next, cells were fixed with 4% (v/v) paraformaldehyde, permeabilized with1% (v/v) Triton X-100. Then, cells were blocked with 1% bovine serum albumin in PBS for 2 hat RT and labeled with rabbit polyclonal anti-GABA (1:100 A2052; Sigma-Aldrich) for 1 hat RT and mouse monoclonal anti-AR (1:50 ab9474; ABCAM) overnight at 4 °C in PBS. The anti-GABA antibody was detected with an Alexa Fluor 488 donkey anti-rabbit antibody (Invitrogen), and the anti-AR antibody was detected with an Alexa Fluor 568 anti-mouse antibody (Invitrogen), incubated for 1 h. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Immunohistochemistry was observed using a Confocal Microscope (Zeiss LSM 510 Axiovert 200 M Laser Scanning).
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