Dako lsab system hrp

The dissected brains were fixed in Carnoy liquid (1 h) and then washed with absolute ethanol (three times within 3 h), absolute ethanol with xylene (1:1) (twice within 1 h), and xylene (3 times within 20 min), and then, after 3-h saturation of tissues with liquid paraffin, the samples were embedded in paraffin blocks. Using a microtome (MICROM HM340E), 3–5 μm serial sections were obtained and placed on silane histological slides (3-aminopropyl-trietoxy-silane, Thermo Scientific, UK). The preparations were deparaffinized in xylene and ethanol with decreasing concentration and then used for further histological staining. In order to expose epitopes, the deparaffinized sections were twice boiled in a microwave oven (700 W, 4 and 3 min) in 10 nM citrate buffer (pH 6.0). Once cooled and washed with phosphate-buffered saline (PBS), the preparations were incubated for 60 min at room temperature with a primary antibody against D1, D2, like used for western blotting analysis, in dilution 1:1000.
In order to visualize the antigen-antibody complex for rabbit antibodies under light microscopy (Axioskop Zeiss, Germany), we used the DAKO LSAB+System-HRP (DakoCytomation, UK), based on the reaction avidin-biotin-horseradish peroxidase with diaminobenzidine (DAB) as chromogen. The result of that reaction was visualized, using diaminobenzidine reaction (DAB, Sigma-Aldrich, Poland).

Tissues fixed in 10% formalin were processed and embedded in paraffin. The blocks were sectioned into 5- to 6-μm slices in a standard rotary microtome and placed on a slide for hematoxylin and eosin staining (H&E).
Anti-VACV primary rabbit polyclonal antibody was used in the IHC at a 1:1000 dilution. The secondary and tertiary antibodies (peroxidase conjugated streptavidin) used were those from the Kit DAKO LSAB + System-HRP (Dako, USA). Two percent bovine serum albumin (BSA) was used for blocking. Ten percent hydrogen peroxide was used to block the endogenous peroxidase. The chromogen used was 3-Amino-9-ethylcarbazole (AEC; Sigma, USA). A teat section from a cow with lesions caused by VACV and lung tissue of mice inoculated intranasally with VACV were used as positive controls. The lung slides were kindly provided by the “Laboratório de Vírus, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG)”. Tissues from the mice in the control group were used as negative controls.
Two scientists read the H&E and IHC stained slides independently. A qualitative analysis of the IHC positive slides was made by giving the average intensity score of 1+, 2+, or 3+, corresponding to focal, multifocal, and diffuse VACV-immunostaining, respectively.

In order to investigate IL1RI expression 10⁴ MSC were seeded per well on 4-well glass slides with polystyrene vessels (BD Falcon, Heidelberg, Germany) in basal medium. After over night adherence, serum-free medium was added 30min before staining. The culture slides were fixed in a 4% formaldehyde solution and MSC were stained with 2 µg/ml of the anti-human IL1RI antibody (AF269, R&D Systems) and the Dako LSAB+ System-HRP (K0690 Dako, Hamburg, Germany) according to the manufacturer’s protocol. Negative controls were stained with the IgG isotype control.

Mouse bone samples and human primary breast cancers and bone metastases were deparaffinised and incubated with 0.07 M citrate buffer (pH 6) for 15 min at 98 °C or processed in acid unmasking buffer for antigen retrieval, then treated with 3% H₂O₂ and incubated overnight at 4 °C with the anti-Notch2 antibody (Santa Cruz Biotechnology, cat: sc5545) or for 1 h at room temperature with the human pan-cytokeratin AE1/AE3 antibody (DAKO, cat: M3515). The staining signals were revealed using the Dako LSAB+ System-HRP (DAKO, cat: k0679). Slides were counterstained with Mayer’s haematoxylin (Sigma-Aldrich, cat: GHS332). Positive and negative controls were performed in parallel.

According to Hsu et al. [51 (link)], sections from the ileocecal region were immunohistochemically stained by cyclin D1 using rabbit monoclonal anti-human cyclin D1 (Cat# 54-0017, CA 94080) (Oncogene, San Francisco, CA, USA) in addition to a detection kit DAKO LSAB^® System HRP (DAB, DAKO, Glostrup, Denmark).