Envision polymer detection system

Additional sections were labeled with anti-clone RAM11 antibody for rabbit
macrophages (Dako, USA). For immunostaining, antigen retrieval was performed with a
Pascal antigen retrieval high-pressure chamber (Dako, Denmark) with 10 mM citrate
buffer, pH 6.0. Endogenous peroxidase activity was blocked by incubation in 3%
hydrogen peroxide, and nonspecific reaction was blocked by incubation in 1% bovine
albumin (Sigma-Aldrich, USA) for 1 h at 37°C. The sections were then incubated
overnight at 4°C with anti-RAM11. Next, the sections were incubated for 30 min at
room temperature with the Envision Polymer Detection System (Dako). The sections were
then incubated with a 3,3′-diamino-benzidine (DAB) chromogen system (Dako) for 2 min
at room temperature and counterstained with hematoxylin. The image analysis of
immunostaining for macrophages was calculated by the percentage of labeled area
relative to total tissue area. The color detection threshold was chosen for the DAB
chromogen (brown staining) in tissue sections. The measurements were performed using
the QWin Image Analysis software (Leica Imaging Systems).

Performed IHC studies of 20 patients with DRE to determine apoptosis by evaluating the presence or absence of active caspase-3. IHC reactions were performed on paraffin-embedded 5–7-μm-thick slices of the brain temporal lobe biopsies according to the standard protocol. Rabbit polyclonal antibody to active caspase-3 (1:100, PC679, Merckmillipore, Darmstadt, Germany) and EnVision polymer detection system (Dako, Santa Clara, CA, USA) were used to detect apoptotic cells. We used active caspase-3 (IHC) to demonstrate apoptosis. Full-length caspase-3 (WB), together with caspase-9, we used to study the pathways of apoptosis. To test for antibodies, staining was performed with the positive control for active caspase-3. Additionally, reactions lacking primary antibodies were done to ensure the specificity of the observed staining.

Immunohistochemical staining of human gastric biopsies for c-Abl and pAbl^T735 was performed on routinely FFPE tissue, using a standardized automated platform (AutostainerPlus, Dako, DN) in combination with Envision polymer detection system (Agilent Technologies, Austria). Details can be found in the Additional file 1.

Antibodies against IL-8 and CXCR2 were purchased from Biorbyt (UK). Anti-CXCR1 was purchased from Boster Bio (USA). Anti-CD3, EnVision polymer detection system, anti-CD68, and Anti-CD34 were purchased from Dako, Agilent Pathology solutions, Denmark. A standard immunohistochemical (IHC) technique was followed as described previously [15 (link)]. A semiquantitative analysis was done using IHC scoring of 0, 1 and 2. A ‘0’ score indicated no scoring, ‘1’ indicated < 30% cells showing immunostaining and ‘2’ indicated > 30% cells showing immunostaining. For correlation study between IL-8, CD68 and CD3 positivity, total percentage positivity was calculated in five different microscopic fields and average percentage was calculated for each case.

After μOCT imaging, the frozen swine coronary arteries were serially sectioned and stained. PM-2K monoclonal antibody (Abcam, Cambridge, UK) was used for the identification of plaque macrophages and α-SMA antibody (Abcam, Cambridge, UK) for SMCs. We used Modified VMT staining technique to highlight elastic fibers and other connective tissue elements. The rabbit arteries were fixed in 10% formalin and processed into paraffin-embedded blocks. Four-micrometer paraffin sections were prepared using a Leica RM2255 microtome (Leica Biosystems). The sections were deparaffinized and rehydrated, and then H&E and von Kossa (CVK-2-IFU, ScyTek Laboratories, West Logan, UT) staining were performed in accordance with the manufacturer’s protocol. For immunohistochemical analysis, antigen retrieval was performed, endogenous peroxidase was blocked; and plaque macrophages were stained using RAM11 primary antibody (M0633, Agilent Dako, Santa Clara, CA). Tissue sections were then labelled with the Envision Polymer Detection System (Agilent Dako).