Dako real envision hrp rabbit mouse

Immunohistochemical detection of β-catenin protein expression in situ was performed in six B3 thymomas and nine thymic carcinoma samples using a rabbit monoclonal antibody against human β-catenin (Cell Signaling, CA, clone 8480s). Immunohistochemistry was performed using a Dako Autostainer (Dako Agilent, Germany). Briefly, 1 μm thick sections of formalin-fixed, paraffin-embedded material were deparaffinized using Xylene and rehydrated in graded ethanol concentrations. Antigen retrieval was performed with an epitope retrieval solution (Cell Signaling, CA) at pH 6.0 for 40 min and cooled down to room temperature for 30 min. Sections were then incubated with a 1:50 dilution in Antibody Diluent (Zytomed ZUCO-25-500) of β-catenin antibody after endogenous peroxidase blocking for 7 min. Sections were incubated with a peroxidase-labeled polymer conjugated to goat anti-mouse or goat anti-rabbit immunoglobulins as a secondary antibody for 30 min (Dako REAL™ EnVision™/HRP, Rabbit/Mouse (ENV)). Staining was visualized with 3, 3’-diaminobenzidine (DAB) as chromogen and slides were counterstained with hematoxylin, dehydrated, and finally mounted. Sections of desmoid tumors showing nuclear β-catenin expression served as a positive control.

The tissue microarray (TMA) dataset (n = 128, Additional file 2: Table S3) was used to evaluate the immune cell infiltration. Following deparaffinization and rehydration, heat-induced epitope retrieval (HIER) was performed by submerging the slides in antigen unmasking solution (Solarbio).
After blocking endogenous peroxidase and nonspecific binding sites (0.3% H2O2 and 5% normal goat serum, sequentially), primary antibodies were applied at 4 °C overnight. Slides were incubated with Dako REAL EnVision HRP rabbit/mouse (belong to K5007, DAKO, Glostrup, Denmark) at RT for 20 min, followed by treatment with Dako REAL DAB + CHROMOGEN and Dako REAL substrate buffer (belong to K5007, DAKO, Glostrup, Denmark) to visualize staining signals under light microscopy, finally counterstained using hematoxylin solution. Stained slides were scanned using Ocus (Grundium, Tampere, Finland) and analyzed with Qupath software (see below).

Embryos from inoculated chicken eggs were collected for gross pathological examination. Histopathological examination and IHC were conducted on selected tissues of the embryos inoculated with viruses of different pathotypes. They were then fixed with 10% NBF, processed, and embedded in paraffin. Sections measuring 4 mm in thickness were prepared and stained with hematoxylin and eosin. Duplicate sections were stained immunohistochemically. IHC staining included the use of primary rabbit anti-NDV polyclonal antibody (Bioss Antibodies, USA) in 1:500 dilution (Dako, S3022), secondary antibody Dako REAL™ envision™/HRP, rabbit/mouse (Dako North America, USA) (K5007), and Dako REAL™ DAB + chromogen in Dako REAL™ Substrate Buffer (Dako North America). After staining, samples were dripped with ultramounted Dako and placed on a hot plate at 70°C for 20 min. Furthermore, slides were mounted, cleared, and sealed with coverslips.