All animals were killed 8 days after treatment. Mouse hearts were harvested and frozen in Optimal Cutting Temperature (OCT) compound (Tissue-Tek, Torrance, CA, USA). Cryo-sections (5 μm thick) were prepared. Masson’s trichrome staining was performed as per manufacturer’s instructions [HT15 Trichrome Staining (Masson) Kit; Sigma-Aldrich]. Fibrosis area was measured by NIH Image J as previously described 23 (link). For immunofluorescence staining, mouse heart cryosections were fixed with 4% PFA, blocked/permeabilized with Protein Block Solution (DAKO) containing 1% saponin (Sigma-Aldrich), and then stained with anti-α-SA (Sigma-Aldrich) and anti-von Willebrand factor (Abcam) antibodies. FITC or Texas-Red secondary antibodies were obtained from Abcam and used in conjunction with these primary antibodies. Images were taken by a Zeiss (Jena, Germany) confocal microscopy system.
Confocal microscopy system
Manufactured by Zeiss
Sourced in Germany
The Zeiss Confocal Microscopy System is a high-resolution imaging tool that uses a laser scanning technique to capture detailed, three-dimensional images of samples. It provides optical sectioning capabilities, allowing for the visualization of specific planes within a specimen.
Automatically generated - may contain errors
Lab products found in correlation
Ab183322, by Abcam (2 mentions)
Nb600 633, by Novus Biologicals (2 mentions)
Hla dr clone l243, by BioLegend (2 mentions)
Ccr2 7a7, by Abcam (2 mentions)
Cd34 q bend1, by Abcam (2 mentions)
Cd64 ab119843, by Abcam (2 mentions)
Biotinconjugated anti mouse or anti rabbit secondary antibodies, by Vector Laboratories (2 mentions)
Abc elite, by Vector Laboratories (2 mentions)
Tunel staining, by Roche (2 mentions)
Protein block solution, by Agilent Technologies (1 mentions)
Anti von willebrand factor, by Abcam (1 mentions)
Ht15 trichrome staining masson kit, by Merck Group (1 mentions)
Fitc or texas red secondary antibodies, by Abcam (1 mentions)
Saponin, by Merck Group (1 mentions)
3 protocols using confocal microscopy system
1
Cardiac Fibrosis Assessment in Mice
2
Multiparametric Analysis of Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Paraffin embedded sections were dewaxed in xylene, rehydrated,
endogenous peroxide activity quenched in 10% methanol and 3%
hydrogen peroxide, processed fro antigen retrieval by boiling in citrate
buffer pH 6.0 containing 0.1% Tween-20, blocked in 1% BSA,
and stained with the following primary antibodies overnight at 4 degrees C:
CD68 (KP1 eBiosceince 1:2000), CCR2 (7A7 Abcam 1:2000), CD34 (Q/bend1 Abcam
1:2000), Collagen 1 (COL-1 Abcam 1:2000), IL-1β (NB600-633 NOVUS
1:2000), Ki67 (ab15580 Abcam 1:1000), CD14 (ab183322 Abcam 1:2000), CD64
(ab119843 Abcam 1:4000), iNOS (ab76198 Abcam 1:1000), HLA-DR (clone L243
Biolegend 1:1000). The primary antibody was detected using a biotin
conjugated anti-mouse or anti-rabbit secondary antibodies (Vector Labs) in
conjunction with streptavidin HRP (ABC Elite, Vector Labs). The PerkinElmer
Opal Multicolor IHC system was utilized to visualize antibody staining per
manufacturer protocol. TUNEL staining (Roche) was performed per
manufacturer’s protocol. Immunofluorescence was visualized on a
Zeiss confocal microscopy system. Macrophages were quantified by examining
at least 4 similarly oriented sections from 4 independent samples in blinded
fashion.
endogenous peroxide activity quenched in 10% methanol and 3%
hydrogen peroxide, processed fro antigen retrieval by boiling in citrate
buffer pH 6.0 containing 0.1% Tween-20, blocked in 1% BSA,
and stained with the following primary antibodies overnight at 4 degrees C:
CD68 (KP1 eBiosceince 1:2000), CCR2 (7A7 Abcam 1:2000), CD34 (Q/bend1 Abcam
1:2000), Collagen 1 (COL-1 Abcam 1:2000), IL-1β (NB600-633 NOVUS
1:2000), Ki67 (ab15580 Abcam 1:1000), CD14 (ab183322 Abcam 1:2000), CD64
(ab119843 Abcam 1:4000), iNOS (ab76198 Abcam 1:1000), HLA-DR (clone L243
Biolegend 1:1000). The primary antibody was detected using a biotin
conjugated anti-mouse or anti-rabbit secondary antibodies (Vector Labs) in
conjunction with streptavidin HRP (ABC Elite, Vector Labs). The PerkinElmer
Opal Multicolor IHC system was utilized to visualize antibody staining per
manufacturer protocol. TUNEL staining (Roche) was performed per
manufacturer’s protocol. Immunofluorescence was visualized on a
Zeiss confocal microscopy system. Macrophages were quantified by examining
at least 4 similarly oriented sections from 4 independent samples in blinded
fashion.
3
Multiparametric Analysis of Tissue Sections
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