Immunohistochemical Analysis of Cardiac Tissues

Tissues were processed as described previously (12 (link)). Briefly, rat hearts were excised after hemodynamic data collection under anesthesia and perfused with 4% paraformaldehyde. Human tissue was fixed with 4% paraformaldehyde. Tissues were cryo-preserved using 30% sucrose and embedded in OCT (TissueTek). Sections were cut to 7µm, using a commercial cryostat, and stained for isolectin B4 (Invitrogen; Carlsbad, CA), α-SMA (Sigma), sarcomeric-α-actin (Santa Cruz Biotechnology; Santa Cruz, CA), anti-human nuclei (Millipore; Billerica, MA), or c-kit⁺ (BD Biosciences) primary antibodies. For Isl-1 staining, the cells were fixed with 4% PFA, permeabilized, overnight incubated with Isl-1 antibody (R & D Systems), incubated with FITC labeled secondary antibody. Cells and tissue sections were counterstained with DAPI (4’,6-diamidino-2-phenylindole) nuclear stain (Sigma; St Louis, MO).

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Sharma S., Mishra R., Simpson D., Wehman B., Colletti E.J., Deshmukh S., Datla S.R., Balachandran K., Guo Y., Chen L., Siddiqui O.T., Kaushal S, & Kaushal S. (2015). Cardiosphere Derived Cells from Pediatric End-Stage Heart Failure Patients Have Enhanced Functional Activity due to the Heat Shock Response Regulating the Secretome. Stem cells (Dayton, Ohio), 33(4), 1213-1229.

Publication 2015

isolectin B4 α-SMA sarcomeric-α-actin anti-human nuclei c-kit+ Isl-1 antibody

Actin Anesthesia Antibodies Antibody C kit Cells Dapi Fitc Hearts Hemodynamic Human Isolectin Nuclei Paraformaldehyde Sarcomeric Stain Sucrose Tissue

Corresponding Organization :

Other organizations : University of Maryland, Baltimore, University of Nevada, Reno, Retina Specialty Institute

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Variable analysis

independent variables

Tissue type (rat hearts vs. human tissue)

dependent variables

Localization and expression of isolectin B4, α-SMA, sarcomeric-α-actin, anti-human nuclei, c-kit+, and Isl-1

control variables

Anesthesia for animal experiments
Perfusion of tissues with 4% paraformaldehyde
Cryopreservation in 30% sucrose and embedding in OCT
Cryosectioning at 7µm thickness
DAPI nuclear staining

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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