Dab peroxidase substrate kit

Tumor microvessels were detected by IHC ofFFPE tumors using a polyclonal antibody to CD31 (ab28364; Abcam) at a dilution of 1:500 for 1 hr at RT in primary antibody diluent (Dako). After washing in Tris-buffered saline containing 0.05% Tween-20 (TBS-T), slides were incubated with biotinylated anti-rabbit labeled polymer (Dako). IHC was visualized using DAB peroxidase substrate kit (Dako) and counterstained with hematoxylin (Sigma-Aldrich). The area of tumor microvessels was quantitatively measured using the ImagePro Plus 4.5 software (Media Cybernetics) as previously described [66 (link)]. Tumor section images were imported into the ImagePro Plus software, where the CD31-positive staining was selected using the color selection function. Positive staining pixels were measured using the area/density (intensity) measurement function and represented as microvessel density.

For immunohistochemistry staining, tissue sections (5 μm thickness) were deparaffinized in xylene and rehydrated through alcohol gradient, incubated with hydrogen peroxide (Dako) for 10 minutes to quench the endogenous peroxidase activity, and then rinsed with PBS three times. Antigen retrieval was performed using a steamer for 30 min in 0.1 M citrate buffer (pH 6.0). Serum-free protein block (Dako) was used to prevent nonspecific protein binding. Sections were then incubated with primary antibodies overnight at 4 °C, followed by a 60-min incubation with anti-rabbit secondary antibody at room temperature. The slides were developed with DAB Peroxidase Substrate Kit (Dako), counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and then mounted with Permount mounting medium (Thermo Fisher Scientific).

To confirm the results of Western blot and immunofluorescence, the other part of the heart tissues was fixed overnight in 4% paraformaldehyde, embedded in paraffin wax, and cut to produce 5-μm–thick sections. For immunohistochemical staining, sections were dewaxed in xylene and rehydrated in serial dilutions of alcohol. Endogenous peroxidase was blocked by immersing the sections in 0.3% H₂O₂ in methanol for 20 minutes at room temperature. The specimens were blocked with 3% nonfat milk for two hours at room temperature, and treated using rabbit polyclonal antibodies against Yap1, β-catenin, and NICD at 4 °C for 12–14 hours. Antigen–antibody complexes were detected using a DAB peroxidase substrate kit according to the manufacturer’s protocol (Dako, Carpinteria, CA, USA). The negative controls were subjected to the same protocol except that PBS was used instead of the primary antibody.

After culture with DEX or cortisol, explants were processed for immunohistochemical analysis as previously described.3, 19 Slides were incubated (overnight at 4°C) with primary antibodies: anti‐mouse BCRP (1:200, BXP‐21, Millipore, Billerica, MA), and anti‐mouse cytokeratin 7 (CK7, 1:500, Dako). Mouse or rabbit IgG1 (Dako) was added instead of primary antibody in controls. After incubation, slides were washed and incubated with the corresponding biotinylated secondary antibody (1:300, 1 hour, Dako). Sections were then washed in PBS (3x for 10′ each time) and incubated with streptavidin‐HRP (30 minutes; Dako). Chromogenic detection of horseradish peroxidase (HRP) activity was achieved by 3,3′‐diaminobenzidine (DAB) reagent (DAB peroxidase substrate kit, Dako). Slides were counterstained with haematoxylin, dehydrated in ascending grades of ethanol and cover slipped. Slides were visualized with an Olympus BX61 upright, motorized microscope with an Olympus DP72 digital camera (Olympus, Tokyo, Japan).

Hematoxylin and eosin staining was carried out following standard protocol. For immunohistochemistry staining, tissue sections (5 μm thickness) were deparaffinized in xylene and rehydrated through alcohol gradient, incubated with hydrogen peroxide (Dako) for 10 minutes to quench the endogenous peroxidase activity, and then rinsed with PBS 3 times. Antigen retrieval was performed using a steamer for 30 minutes in 0.1 M citrate buffer (pH 6.0). Serum-free protein block (Dako) was used to prevent nonspecific protein binding. Sections were then incubated with primary antibodies overnight at 4°C followed by 60-minute incubation with anti-rabbit secondary antibody (111-035-144, Jackson ImmunoResearch) at room temperature. The slides were developed with DAB Peroxidase Substrate Kit (Dako), counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and then mounted with Permount mounting medium (Thermo Fisher Scientific).

Tumor tissues and major organs were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and cut into 5-µm-thick sections. The tissue sections were dewaxed (by heating at 60°C for 2 h), washed in xylene, and then rehydrated using a graded ethanol series. After antigen retrieval using microwave heating and blocking with 20% goat serum, the tissue sections were incubated with primary antibody (anti-Ki67 antibody; ab15580; Abcam) overnight at 4°C. Subsequently, immunodetection was carried out using a 3,3’-diaminobenzidine (DAB) peroxidase substrate kit (K5007; Dako) according to the manufacturer’s protocol. For HE staining, the slides were stained with hematoxylin and eosin sequentially after dewaxing and rehydration.

Explants were processed for immunohistochemical analysis as previously described [10 (link),22 (link)]. Briefly, slides were deparaffinized, rehydrated, and subjected to antigen retrieval with sodium citrate. After blocking with Dako protein block (Dako, Mississauga, ON, Canada), the slides were incubated overnight at 4 °C with primary antibodies: anti-mouse BCRP (1:200, BXP-21, ab3380, Millipore, Billerica, MA, USA), anti-mouse HLA-G (1:300, 4H84, Exbio, Burlington, ON, Canada), and anti-mouse CK7 (1:1000; M7018, Dako). In the controls, mouse or rabbit IgG1 (Dako) was added instead of the primary antibody. After incubation, the slides were washed and incubated with the corresponding biotinylated secondary antibody (1:300, 1 h, Dako). Sections were washed in 1× PBS (3 × 10 min) and incubated with streptavidin-HRP (30 min; Dako); immunostaining was detected with the peroxidase substrate kit DAB (Dako). The slides were counterstained with hematoxylin, dehydrated in ascending concentrations of ethanol, and cover slipped with mounting medium. Visualization was undertaken with an Olympus BX61 upright, motorized microscope, and representative images were captured using an Olympus DP72 digital camera (Olympus, Tokyo, Japan).