Envision dual link system hrp kit

Formalin-fixed paraffin-embedded (FFPE) tumor tissues from each of the treated mice were placed on glass slides. The tissues were deparaffinized and hydrated through xylene and a graded alcohol series. This was followed by permeabilization with 0.1% Triton X-100 in PBS, antigen retrieval with 10 mM sodium citrate buffer, and quenching of endogenous peroxidase activity with 0.3% H₂O₂. 5% BSA was used to block the tissues for 1 h at room temperature. Primary antibodies against SPHK2 (antibody: D2V3G) and the serine proteases [trypsin 1 and 3 (PRSS1, PRSS3), and putative trypsin 6 (PRSS3P2)] (antibody Abcam 200997) were used at concentrations of 1:500 and 1:1000 respectively, incubating overnight. The EnVision+ Dual Link System-HRP kit (Dako) was used as a secondary antibody. DAB Substrate Kit, Peroxidase (Vector Laboratories) was used to precipitate, at the location of the HRP, which was later visualized using light microscopy at 20× and 40× magnification.

Human NSCLC tissues were stained with IHC using the EnVision™ + Dual Link System-HRP Kit (Dako; Carpinteria, CA). The rabbit monoclonal antibody against DR4 (D9S1R; #42533) was purchased from Cell Signaling Technology (Danvers, MA 01923) and used at 1:100 dilutions overnight at 4^oC. The specificity of the antibody was determined with matched IgG isotype antibody as a negative control in IHC. Moreover, a single band of correct molecular weight in Western blotting was assured. Both the percentage of positive staining in tumor cells and intensity of staining were scored. The intensity of IHC staining was measured by using a numerical scale (0 = no expression, 1 = weak expression, 2 = moderate expression and 3 = strong expression). The staining data were finally quantified as the weighted index (WI) (WI = % positive staining in tumor x intensity score) as previously described 54 (link). DR4 staining was scored as negative (≤ 10 WI) and positive staining (> 10 WI), respectively. The WI was determined by 2 individuals, and the final values were the average of the two readings. cPARP in xenograft tissues were stained with cPARP antibody purchased from Cell Signaling Technology (#5625) at 1:50 dilution.

On day 28, the prepared slides (as above‐mentioned) were used for Immunohistochemistry (IHC) analysis of GFAP positive astrocytes. In brief, the endogenous activity was inhibited by using 3% H₂O₂ for 20 min after antigen retrieval. The slides were exposed to anti‐GFAP and‐von Willebrand factor (vWF) antibodies (Dako) for 1 h at room temperature and washed three times with PBS. The procedure was continued with the addition of the EnVision + Dual Link System HRP kit (Dako). DAB was exploited as chromagen. The number of GFAP‐positive astrocytes and vWF‐positive endothelial cells were calculated in the periphery of the injured area in different groups and compared to the control stroke mice. To this end, we took serial images with high magnification from the periphery of the injured area of each slide. Then, the periphery zones were randomly outlined into five subfields and the number of vWF‐ and GFAP‐positive cells were counted and compared to the control group.

The angiogenesis potential of Mel- and CD144⁺ ECs-loaded Alg + Fib hydrogel was investigated using IHC staining. For this purpose, paraffin-embedded blocks were cut into 5-µm thick slides and rehydrated in ascending concentrations of alcohols. The endogenous peroxidase activity was inhibited using 3% (v/v) H₂O₂. The antigen retrieval process was performed by the incubation of slides in boiling sodium citrate buffer with a pH value of 6 for 20 min. The procedure was followed by overnight incubation samples with anti-α-SMA (dilution: 1:100; Dako, Denmark) and anti-vWF (dilution: 1:100; Dako, Denmark) antibodies. After three washes with PBS, an HRP-conjugated secondary antibody (EnVision + Dual Link System HRP kit; Dako) with DAB was used to visualize the immunoreactive foci. For semi-quantification analysis, 10 high-power fields were randomly examined in each slide and the mean vWF⁺ ECs and α-SMA⁺ vessels were compared to the control hydrogel-free frozen group (FT).

Immunohistochemistry staining were performed as previously described [22 (link)]. For each patient, we selected the most relevant tumor paraffin blocks from interval debulking surgery. Paraffin-embedded sections were deparaffinized in xylene and rehydrated in graded alcohol. Immunostaining was perfomed manually, using the Dako Envision + Dual Link System-HRP kit and anti-IL8 primary antibodies (mouse monoclonal to IL8, clone 807, ref. Ab18672, Abcam, UK). Immunostaining specificity was verified with a control antibody and a positive tissue control. All slides were counterstained with hematoxylin. Microscopic analyses were performed using a Nikon Eclipse 90i microscope (Nikon, Nikon Instrument B.V., France). Representative photographs (20× magnification) of tumor immunostaining were performed using the NIS-Element BR software package (Nikon, Nikon Instrument B.V., France). Standardized quantitative analysis of IL8 immunostaining was based on Image J software (National Institutes of health, USA). Briefly, a grid delimitating 10,000 μm² squares was applied to each photograph, and 3 randomly selected squares were analyzed. We used the Image J cell counter plugin to count the stained an unstained tumor cells within each square. IL8 expression was defined as the rate of stained tumor cells.