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.