Envision flex dab chromogen

Mice were immersion fixed in 4% PFA rather than perfused because of their small size, brains embedded in paraffin and 7 μm sections obtained as described (Deters et al., 2008 (link)). For antigen retrieval, the sections were microwaved in AR buffer (Dako Envision Flex Target Retrieval Solution low pH #K8005) for 15 min on low power before the buffer started to boil. The sections were then left to cool for 1h in the AR buffer at RT. Blocking was done in 20% FBS, 1% BSA in TBST for 1h at RT. Primary antibodies were used over night at 4°C and secondary antibodies for 1.5h at RT. Primary antibodies were Myc-Tag rabbit mAb (Cell Signaling Technologies, #71D10, used at 1:100) and anti-Human PHF-Tau Monoclonal Antibody AT8 (Thermo Fisher, MN1020, used at 1:400). Secondary antibodies were polyclonal goat anti-rabbit IgG biotinylated (Dako, #E0432, used at 1:500) and polyclonal rabbit anti-mouse IgG biotinylated (Dako, #E0413, used at 1:500). The VectaStain Elite ABC Kit #PK6102 was used and Envision Flex DAB chromogen (Dako, #DM827) and Envision Flex Substrate Buffer (Dako, # DM823) for DAB development. Haematoxylin was used for counter-staining.

Our previous work described the immunohistochemical stainings of CD3, CD8, Ki67, and cytokeratin used to determine the phenotypic subgroups [31 (link)].
The immunohistochemistry of the angiogenic growth factors VEGF, bFGF, and PDGF-bb was carried out using a similar method used to describe Ki67 and cytokeratin stainings [31 (link)]. First, slides were pretreated for 15 min with an EnVision Flex target retrieval solution (Dako, Santa Clara, CA, USA, DM828) at 98 °C in a pretreatment module (Agilent Technologies Inc., Dako, Santa Clara, CA, USA). Next, the pretreated slides were incubated with primary antibodies (mouse polyclonal anti-human VEGF (PharMingen, San Diego, CA, USA, diluted to 1:100), rabbit polyclonal anti-human bFGF (Bioss, Boston, Massachusetts, USA, diluted to 1:800), and rabbit polyclonal anti-human PDGF-bb (Thermo Scientific, Waltham, Massachusetts, USA, diluted to 1:500) in an Autostainer 480 (Lab Vision Corp, Fermont, CA, USA) overnight at room temperature. Subsequently, the slides were treated for 20 min with HRP-labeled EnVision Flex/HRP secondary antibodies (Dako, Santa Clara, CA, USA, SM802), which were then visualized by 10 min incubation with EnVision Flex DAB chromogen (Dako, Santa Clara, CA, USA, DM827). Finally, the slides were counterstained with Meyer’s hematoxylin and washed in tap water.

Histological sections were cut from formalin-fixed, paraffin-embedded tissues (4-μm), de-waxed and dehydrated in xylene and ethanol, respectively [47 (link)]. Tissues were incubated in methanol and hydrogen peroxide (3%) for 12 min. Sections were incubated with primary anti-FABP9 antibody (1:500 sheep polyclonal FABP9 antibody, R&D Company) or with anti-FAB6 antibody (1:50 rabbit polyclonal FABP6 antibody, Sigma-Aldrich, UK) at room temperature for 1 hour and then incubated with a rabbit anti-sheep IgG linker or mouse anti-rabbit IgG linker (Vector Laboratories, Burlingame, CA, USA) for 30 min. Sections were incubated with Envision™ FLEX DAB+ chromogen mixed with Envision™ FLEX substrate (ldop/ml) (Dakocytomation, Ely, UK) for 15min. All sections were counterstained with hematoxylin, mounted on glass slides with cover slip using DPX mountant, and observed by light microscope for scoring.