Envision detection system peroxidase dab kit

A human prostate cancer TMA (PR 956a) was purchased from US Biomax (Rockville, MD). A TMA plate contains tissue samples from 8 patients with normal prostates and 36 patients with prostate cancer, and one or two spotted tissues of each patient are placed on the array. IHC staining was performed using the EnVision Detection System Peroxidase/DAB+ kit (K5007) according to the manufacturer's protocol (Dako, Glostrup, Denmark) and the method described by Shin et al. [32 (link)]. Tissue specimens were stained with rabbit polyclonal antibody against CCR2 and goat polyclonal antibody against CCR4. Microwave antigen retrieval was performed in Dako Target Retrieval Solution for 10 min. The arrays were analyzed by a blinded pathologist, and the intensities of the stained epithelial cells were recorded. Staining intensity was scored on a scale from – to +++ (–, negative; +, weak; ++, moderate; +++, strong intensity staining).

Rats were euthanized 3 weeks after cells or vehicle transplantation by intravenously injection of phenobarbital and phenytoin (Beuthanasia-D). The hearts were harvested, rinsed in non-sterile saline, and perfused with 0.9% saline and 4% paraformaldehyde sequentially. The right and left atria and the right ventricle were harvested. LV was sliced parallel to the short axis at 1-mm thickness. Picrosirius red/fast green staining was performed to determine the infarct/fibrotic area. Infarct size was calculated as Σ (infarct area/block area) and expressed as a percentage of the left ventricle. The hAFSC-iPSC-CM graft was identified by immunofluorescence staining with primary antibodies directed against α-actinin and human-specific HLA-ABC (Table S2) and the fluorescent secondary antibodies (Alexa-conjugated, species-specific antibodies, Table S2). Graft size was determined as Σ (graft area/infarct area) and expressed as a percentage of the infarct region.
To check post-transplant immune reaction, we performed immunohistochemistry staining with primary antibodies against CD3, CD20 and CD68 (Table S2) and EnVision Detection System, Peroxidase/DAB kit (DAKO, K5007), followed by the HRP secondary antibody. Images were acquired using an Olympus BX51 microscope (OLYMPUS, Tokyo, Japan) and quantitated using Image J software.