Tissue analysis (online supplement)
1. Early Time-Point (Day 6 Post-MI)
Tissue Preparation:
Hearts were arrested in diastole with intravenous injection of saturated potassium chloride, and
the hearts were removed. The hearts were then perfused with 10% formalin injected into the
aorta. When successful fixation had been achieved, the hearts were immersed in 10% formalin
for 12 hours. They were then transferred to 80 % alcohol and then paraffin embedded. Paraffin
embedded sections from the mid-papillary level of the left ventricle were examined as follows.
Histological Detection of GFP:
Two sets of paraffin embedded tissue were deparaffinized in changes of xylene and rehydrated in
decreasing concentrations of ethanol. The sections were then treated with trypsin. After rinsing
in distilled water, the sections were treated with a peroxidase solution (Peroxidazed, Biocare,
Concord, CA) to quench endogenous peroxides. The sections were rinsed with phosphate
buffered saline (PBS) and then a universal block (Biocare). A rabbit antibody against Green
Fluorescent Protein (Invitrogen/Molecular Probes, Eugene, OR, 1: 200 dilution) was applied to
sections for 90 minutes at room temperature. After washing with PBS for 15 minutes, a Rabbit
on Rodent secondary polymer (Biocare) was applied to the sections for 30 minutes at room
temperature. The stain was then developed using a DAB chromogen (Biocare). One set was
stained with antibodies against mouse anti-troponin-I (Abcam, Cambridge, MA; 1:50 dilution) as
a second marker using the same protocol as the other troponin stains conducted, and the other set
was stained for alpha smooth muscle actin (Sigma-Aldrich, St. Louis, MO; 1:100 dilution) using
the same protocol as the other smooth muscle staining. Entire sections were examined for the colocalization of GFP and troponin-I or SMA within the same cell.
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Histological Detection of Capillaries and Arterioles:
Paraffin-embedded tissues were deparaffinized in changes of xylene and rehydrated in
decreasing concentrations of ethanol. Sections were treated with trypsin. After rinsing in
distilled water, the sections were treated with a peroxidase solution (Biocare). The sections were
rinsed with Tris Buffered Solution (TBS) and then a universal block (Biocare). A primary rat
antibody against CD31 (Biocare, 1:25 dilution) was applied to the sections for 2 hours at room
temperature. The slides were then rinsed with TBS. A Rat Detection Kit (Biocare) was used to
detect the rat-anti-CD31 antibody. The stain was developed using DAB. A denaturing solution
(Biocare) was applied for 3 minutes to stop any reaction from the CD31 stain, before proceeding
to stain for smooth muscle actin. After washing the sections in TBS, a mouse primary antibody
against alpha-smooth musclactin (Sigma-Aldrich, St. Louis, MO; 1:100 dilution) was applied for
one hour at room temperature The tissues were washed with TBS and a Mouse on Mouse
Polymer (Biocare) conjugated to alkaline phosphatase was applied to the sections for 20 minutes
at room temperature. The sections were washed in TBS again, and the stain was developed using
a Ferangi Blue Chromogen Kit (Biocare). The tissues were then dehydrated in increasing
concentrations of ethanol, and then 3 changes of xylene. The negative controls were treated
using the same methodology, omitting the primary antibody, and replacing it with the diluent.
Low power photomicrographs were taken of three regions: Infarct Zone (IZ), Border Zone (BZ)
and Remote Myocardium (RM). These images were analyzed using Image Pro software. The
software was taught to recognize brown CD31+ staining using the Magic Wand tool and the
recognition parameters were modified for each image until representative selection was
achieved, by an investigator blinded to the identity of the image. A percentage vessel area was
obtained for each of the three regions. The absolute number of arterioles was counted manually
in 5 high power fields within each region.
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Histological Detection of Ki67+ Cardiomyocytes:
Paraffin embedded tissues were deparaffinized in changes of xylene and rehydrated in decreasing
concentrations of ethanol.
Antigen retrieval was then performed in citrate buffer solution
(Biocare). Sections were then rinsed with distilled water, and treated with peroxidase, TBS, and
universal block as described above. A primary rat monoclonal antibody against Ki67 (DAKO,
Capinteria, CA; 1:10 dilution) was applied to the sections for 2 hours at room temperature. After
washing with TBS, a Rat Detection Kit (Biocare) was used to detect the primary antibody. The
stain was developed using DAB. The sections were then denatured for 5 minutes using
denaturing solution as above. After blocking with a universal block (Biocare) for 20 minutes, a
mouse anti-Troponin-I antibody (Abcam, Cambridge, MA) was diluted in TBS and applied to
sections for 1.5 hours at room temperature. Sections were rinsed with TBS and a Mouse on
Mouse Polymer (Biocare), conjugated to alkaline phosphatase, was applied to sections for 20
minutes. After rinsing sections, the stain was developed using Vulcan Fast Red Chromogen and
counterstained with CAT hematoxylin (both from Biocare).
The number of Ki67+
cardiomyocytes was counted in five high powerfields per region (IZ, BZ and RM) by two
blinded counters. These numbers were then averaged. A positive Ki67+ cardiomyocyte was
defined as one with a positively stained nucleus, morphologically resembling a cardiomyocyte
nucleus completely surrounded by troponin staining.
Histological Detection of PCNA+ Cardiomyocytes:
Paraffin embedded tissues were deparaffinized, rehydrated, subjected to antigen retrieval, treated
with peroxidase, TBS, and universal block as described above for Ki67 staining. A primary
mouse antibody against PCNA (Biocare, 1:200 dilution) was applied to the sections for 2 hours
at room temperature. After washing with TBS, a Mouse on Mouse polymer conjugated to
alkaline phosphatase (Biocare) was used to detect the primary antibody. The stain was developed
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using Ferangi Blue. The sections were then washed with distilled water. After blocking with
universal block, troponin-I staining using Vulcan Fast Red was carried out as described above
for Ki67 staining. The number of PCNA+ cardiomyocytes was counted blinded in five high
powerfields per region (IZ, BZ and RM). These numbers were then averaged. A positive
PCNA+ cardiomyocyte was defined as a one with a positively stained nucleus, morphologically
resembling a cardiomyocyte nucleus completely surrounded by troponin staining.
Histological detection of apoptotic cardiomyocytes:
5 μm sections at mid-papillary level were taken from paraffin-embedded hearts harvested 6 days
post-MI. Apoptotic cardiomyoctes were identified using two methods: TUNEL co-stained for
troponin-I (TnI) and active caspase-3 co-stained with TnI. TUNEL staining was performed with
ApopTag® Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA)
according to manufacturer’s protocol, except the terminal deoxynucleotidyl transferase was
diluted to 50% in the supplied reaction buffer. After color development in DAB, denature
solution was applied (Biocare) to prevent cross reaction with the following TnI staining. The
slides were treated with Rodent Block M (Biocare) to block endogenous IgG, and then incubated
for 1.5 hour with TnI primary antibody (Abcam, 1:50) dissolved in TBS. For detection of TnI
primary antibodies, mouse-on-mouse alkaline phosphatase polymer (Biocare) was applied to the
slides for 25 minutes at room temperature, followed by color development in Vulcan Fast Red.
Finally, slides were counterstained with hematoxylin. TUNEL-positive cardiomyocytes were
defined by the presence of both DAB nuclear staining and cardiomyocyte nucleus morphology
that was completely surrounded by troponin-I staining. For fluorescent detection of active
caspase-3, sections were deparaffinized and antigen-retrieved in Rodent Decloaker (Biocare,
Concord, CA) by 20 minutes of microwave heating. The slides were treated with Rodent Block
M to block endogenous IgG, followed by overnight 4°C incubation in staining buffer containing
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both rabbit anti-mouse cleaved-caspase-3 antibody (BD Pharmingen, San Jose, CA, 1:100) and
mouse anti-mouse TnI antibody (Abcam, 1:50). The slides were then incubated in secondary
goat anti-rabbit IgG conjugated to Alexa Fluor® 488 and secondary goat anti-mouse IgG
conjugated to Alexa Fluor® 546 (Invitrogen, Carlsbad, CA), followed by preservation in
Vectashield® mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). High
power field pictures at border zone were taken for quantification of caspase-positive
cardiomyocytes, defined by presence of caspase-3 signal completely enclosed in cells with TnI
staining.
2. Late Time-Point (Day 28 post-MI)
Tissue Preparation:
Hearts were arrested in diastole with intravenous injection of saturated potassium chloride, and
the hearts were removed. They were then frozen in OCT for tissue sectioning using a cryostat.
Histological Detection of GFP:
GFP was detected in tissue sections by both direct intrinsic fluorescence and by immunostaining.
Frozen sections were incubated for 1 hour at room temperature in antibody buffer containing
rabbit polyclonal antibody against GFP (Invitrogen/Molecular Probes, 1:1000 dilution), followed
by a secondary goat anti-rabbit IgG conjugated to Alexa Fluor 660 (far red emission), followed
by a tertiary donkey anti-goat IgG conjugated to Alexa Fluor 488 (green emission) for further
amplification of the green signal. Red fluorophores were avoided, so that true GFP could be
distinguished from autofluorescence by bright signal in the green and far red emission
wavelengths with no signal in the intervening red emission wavelength. Negative staining
controls lacking primary antibody were always performed. Sections from animals that did not
receive GFP-expressing cells were included to control for fluorescent artifacts that could be
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misinterpreted as GFP fluorescence. For colorimetric detection of GFP, the secondary antibody
consisted of an HRP-conjugated goat anti-rabbit IgG. Endogenous peroxidase was quenched
with 3% hydrogen peroxide in methanol for 30 min. Sections were then immersed in antigen
retrieval solution (Vector Laboratories Inc., Burlingame, CA) and boiled in a microwave oven
for 30 minutes and blocked with normal goat serum blocking buffer (ImmunoVision
Technologies Co., Brisbane, CA) for 60 minutes. The slides were incubated overnight at 4ºC in
normal goat serum blocking buffer containing a rabbit polyclonal antibody against GFP
(Molecular Probes, 1:1000 dilution). Sections were rinsed in normal goat serum blocking buffer,
and then incubated for 30 minutes in a HRP-conjugated goat anti-rabbit IgG (ImmunoVision
Technologies Co.). Color was developed with DAB. A negative staining control lacking
primary antibody was performed.
Immunofluorescence (non-GFP) and lectin staining:
Frozen tissue sections were fixed with 1.5% formaldehyde for 15 minutes and blocked with
antibody buffer consisting of 2% normal goat serum, 0.3% Triton X-100 and 0.02% sodium
azide in PBS for 30 minutes. When mouse primary antibodies were to be used, an additional
blocking step was added in which sections were incubated for 30 minutes in a mouse-on-mouse
blocking solution based on the method of Lu and Partridge30 consisting 0.2 mg/ml AffiniPure
Fab fragment goat anti-mouse IgG (H+L), 0.2 mg/ml AffiniPure Fab fragment goat anti-rat IgG
(H+L), and 0.2 mg/ml ChromePure goat IgG, Fc fragment (all from Jackson ImmunoResearch
Laboratories, West Grove, PA) in antibody buffer. The slides were incubated for 1 hour at room
temperature in antibody buffer containing individual or multiple primary antibodies as follows: a
rat monoclonal antibody against mouse F4/80 (BD Pharmingen, San Diego, CA; 1:100 dilution),
a rabbit polyclonal antibody against human cardiac troponin T (ab10224, Abcam, Cambridge,
MA; reacts with mouse; 1:200), and mouse monoclonal antibodies against the following
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markers: α-smooth muscle actin (clone 1A4; ICN Biomedicals, Aurora, OH; 1:400 dilution),
cardiac troponin I [4C2] (ab10231, Abcam; 1:200), the pericyte marker NG2 (clone 132.38;
Chemicon, Temecula, CA; 1:200), CD45 (BD, Franklin Lakes, NJ; 1:500), and human NuMA
(nuclear mitotic apparatus antigen, Abcam, 1:50). The troponin T antibody was initially used for
cardiomyocyte staining, but tissue autofluorescence was sufficient for visualization of
cardiomyocytes for most needs. Sections were rinsed in antibody buffer, and then incubated for
1 hour in the appropriate secondary anti-IgG conjugated to Alexa Fluor 350, Alexa Fluor 488,
Alexa Fluor 546, or Alexa Fluor 660 (Invitrogen/Molecular Probes, Carlsbad, CA, 1:200
dilution), with Hoechst 33258 nuclear dye at 0.1 µg/ml when appropriate. For day 28 capillary
staining, sections were stained as described above but biotinylated BS-1 isolectin B4 from
Griffonia simplicifolia was used in place of the primary antibody (Sigma-Aldrich, 1:100
dilution), and streptavidin conjugated to Alexa Fluor 647 (Invitrogen/Molecular Probes, 1:100
dilution) was used for detection. The slides were then rinsed, mounted, and viewed with a Nikon
E800 fluorescence microscope using Openlab software (Improvision, Lexington, MA). Negative
staining controls lacking primary antibody were always performed.
Manual quantitation of blood vessels at day 28 post-implantation:
Capillaries were recognized by BS1 isolectin B4 staining of endothelial cells in one color, and
arterioles were detected by α-smooth muscle actin immunofluorescence in a different color.
Digital photomicrographs of stained vessels were obtained with Openlab software. Capillary
segment density and arteriole density indices were calculated by counting the number of distinct
vessel segments in high-magnification fields.
Vessel area density and vessel length density indices were calculated as follows. A pattern
consisting of repeating sine waves in a third color was imported into Openlab as a new layer,
merged with each capillary photo, and the number of intersections of vessels with the pattern was
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counted. Vessel area density index was defined as the total number of intersections of capillaries
and overlay pattern.
Vessel length density was defined as the number of intersections of
capillary centerlines and overlay pattern. To control for artificially decreased numbers due to
rips and gaps in the tissue, the number of sine wave peaks and troughs that occurred within tissue
was counted for each field, and all vessel density indices were normalized to the number of
tissue sine wave peaks/troughs to yield corrected indices for each field.
performed by an investigator blinded to the identity of the sections.
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All counts were