Cells cultured in vitro were fixed in 4% paraformaldehyde at room temperature for 25 min or heart tissues were dissected and fixed in 4% paraformaldehyde at 4°C overnight. The fixed heart tissues were washed three times with PBS and equilibrated in 30% sucrose at 4°C for 2 days before freezing and cryosectioning. Eight micrometer frozen sections were prepared. Cells or tissue sections were blocked with 5% BSA and 5% goat serum and then stained with the respective primary antibodies at 10 ug/ml at 4˚C overnight. Anti-mouse primary antibodies used: COLA1 (abcam, ab34710), cTnT (abcam, ab8295), pH3 (Millipore, 06-570), Ki67 (abcam, ab15580) or cCASP3 (Cell signaling technology, 9661). Alexa-Fluor-488- or Alexa-Fluor-546-conjugated secondary antibodies (Invitrogen) were used at room temperature for 30 min in the dark. For cell death analysis, an in situ cell death detection kit by TUNEL was also used per manufacturer's instruction (Roche, 1256792910). Slides were mounted with DAPI-containing fluorescence mounting medium (Dako) and fluorescence was detected with an upright fluorescence microscope, inverted fluorescence microscope or confocal microscope (all Leica). Images were processed with the ImageJ software and cTNT coverage was analyzed based on this formula: cTNT+ area/total area.
Dapi containing fluorescence mounting medium
Manufactured by Agilent Technologies
DAPI-containing fluorescence mounting medium is a reagent used to preserve and protect fluorescent-labeled samples for microscopy analysis. It provides a refractive index-matched solution to mount the sample and maintain the fluorescence signal. The DAPI component stains nucleic acids, allowing for the visualization of cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Confocal microscope, by Leica (4 mentions)
Upright fluorescence microscope, by Leica (3 mentions)
Inverted fluorescence microscope, by Leica (3 mentions)
Alexa fluor 488 or alexa fluor 546 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ab15580, by Cell Signaling Technology (1 mentions)
C casp3, by Cell Signaling Technology (1 mentions)
Ab34710, by Abcam (1 mentions)
Aurora b, by Abcam (1 mentions)
Cola1, by Abcam (1 mentions)
Isolectin b4, by Vector Laboratories (1 mentions)
α sma, by Agilent Technologies (1 mentions)
Alexa fluor 546 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab6671, by Abcam (1 mentions)
Mab1435, by Merck Group (1 mentions)
A32723, by Thermo Fisher Scientific (1 mentions)
A32731, by Thermo Fisher Scientific (1 mentions)
Ab15323, by Abcam (1 mentions)
Glucagon, by Abcam (1 mentions)
Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using dapi containing fluorescence mounting medium
1
Immunostaining of Cardiac Tissue
2
Immunostaining and Quantification of Cardiac Tissue
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Heart tissues were dissected and fixed in 4% paraformaldehyde at 4°C overnight. The fixed tissues were washed three times with PBS and equilibrated in 30% sucrose at 4°C for 2 days before freezing and cryosectioning. Eight micrometer frozen sections were blocked at 2% goat serum and then stained with the respective primary antibodies at 10 ug/ml at 4˚C overnight. Anti-mouse primary antibodies used: COLA1 (abcam, ab34710), cTnT (abcam, ab8295), Ki67 (eBiosciences 14-5698-82), pH3 (Millipore, 06-570) and Aurora B (abcam, ab2254). Anti-human primary antibodies used: cTnT (RnD systems, MAB1874), Ki67 (abcam, ab15580) and Aurora B (abcam, ab2254). Alexa-Fluor-488- or Alexa-Fluor-546-conjugated secondary antibodies (Invitrogen) were used at room temperature for 30 minutes in the dark. Slides were mounted with DAPI-containing fluorescence mounting medium (Dako) and fluorescence was detected with an upright fluorescence microscope, inverted fluorescence microscope or confocal microscope (all Leica). Images were processed with the ImageJ software and cTNT coverage was analyzed based on this formula: cTnT+ area/total area. For blind cell count of proliferating cardiomyocytes, contaminating cell types without cTnT expression and background staining without DAPI+ nuclei were excluded.
3
Immunofluorescence Staining of Frozen Tissue Sections
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The fixed tissues were washed three times with PBS and equilibrated in 30% sucrose at 4°C for 2 days before freezing and cryosectioning. Eight micrometer frozen sections were prepared; and were blocked with 5% BSA and 5% goat serum. After that, the sections were stained with the respective primary antibodies at 10 μg/ml at 4°C overnight. Anti-mouse primary antibodies used: CD31 (Biolegend, Cat. No. 102501), isolectin B4 (Vector Laboratories, Cat. No. FL-1201-0.5) or α-SMA (Dako, Cat. No. M085101-2). Alexa-Fluor-488- or Alexa-Fluor-546-conjugated secondary antibodies (Invitrogen) were used at room temperature for 30 min in the dark. Slides were mounted with DAPI-containing fluorescence mounting medium (Dako) and fluorescence was detected with an upright fluorescence microscope, inverted fluorescence microscope or confocal microscope (all Leica). Images were processed with the ImageJ software.
4
Immunohistochemical Analysis of Macrophage Markers
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Next, 5-µm-thick transverse sections were blocked with PBS containing 1% BSA, 3% normal goat serum, and 0.4% Triton X-100 for 1 h at room temperature following the antigen retrieval process. Tissues were incubated at 4 °C in a humidified chamber overnight with primary antibodies anti-ED-1 (1:200; MAB1435; Merck, Kenilworth, NJ, USA), iNOS (1:200; #ab15323, Abcam, Cambridge, UK), and TNF-α1 (200; #ab6671; Abcam). Alexa Fluor 488-labeled goat anti-mouse-IgG (1:1000; #A32723, Invitrogen, Carlsbad, CA, USA), and Alexa Fluor 633-labeled goat anti-rabbit-IgG (1:1000; #A32731, Invitrogen) were used as secondary antibodies. After immunolabeling, the tissue slides were mounted in DAPI-containing fluorescence mounting medium (Dako).
5
Immunofluorescence Analysis of Kidney Grafts
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Kidney grafts were dissected and fixed in 4% paraformaldehyde at 4 °C overnight. The fixed grafts were washed three times with PBS and equilibrated in 30% sucrose for 2 days before freezing and cryosectioning. Six-micrometer sections were blocked at 2% goat serum and then stained with the respective primary antibodies at 10 μg/ml at 4 °C overnight. Anti-human primary antibodies used are the following: PDX1 (R&D systems), NKX6.1 (R&D systems), GLUCAGON (Abcam), and C-PEPTIDE (DSHB), and the anti-mouse primary antibodies used are the following: Ki67 (eBiosciences) and FOXP3 (Cell Signaling Technology). Alexa-Fluor-488- or Alexa-Fluor-594-conjugated secondary antibodies (Invitrogen) were used at room temperature for 30 min in the dark. Slides were mounted with DAPI-containing fluorescence mounting medium (Dako), and fluorescence was detected with a confocal microscope (Leica). Some sections were also stained with hematoxylin and eosin (H&E) for histological analyses.
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