The paraffin‐embedded sections were rehydrated in a series of xylene and ethanol baths. Antigen retrieval was achieved by using a citrate buffer (0.01 m , pH 6.0, 0.05% tween) for CYP17 or Tris/EDTA (10 mm Tris base, 1 mm EDTA, 0.05% tween, pH 9.0) for Wnt4 at 98°C for 10 minutes. The slides were immersed in 3.5% (CYP17) or 0.35% (Wnt4) H2O2 in Tris buffered saline with 0.1% tween (TBST0.1%, 0.02 m , pH 7.6) for 30 minutes to block endogenous peroxidase; 5–10% normal goat serum (NGS) in 1% bovine serum albumin (BSA) was applied to the slides to block nonspecific binding sites. The slides were incubated with the anti‐CYP17 antibody (courtesy of A.J. Conley, rabbit‐anti‐bovine, 1 : 7,500) or the anti‐Wnt4 antibody (rabbit‐anti‐human, 1 : 6,000) and kept at 4°C overnight. The following day, secondary anti‐rabbit antibody (Envision, K4003) was applied for 50–90 minutes. Bound antibody was visualized with the DAB substrate kit for peroxidase (DAKO, K3468, Heverlee, Belgium), after which the slides were counterstained with hematoxylin, dehydrated in a series of ethanol and xylene baths, and embedded with VectaMount Mounting Medium (H‐5000).
Dab substrate kit for peroxidase
Manufactured by Agilent Technologies
Sourced in Belgium, United States
The DAB substrate kit for peroxidase is a laboratory product used for the detection and visualization of peroxidase enzyme activity in various biological samples. The kit provides the necessary reagents for the chromogenic detection of peroxidase using 3,3'-diaminobenzidine (DAB) as the substrate. The core function of this product is to enable the specific localization and quantification of peroxidase-labeled targets in research and diagnostic applications.
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2 protocols using dab substrate kit for peroxidase
1
Immunohistochemical Analysis of CYP17 and Wnt4
2
Quantitative Analysis of Epidermal Thickness and Cytokine Expression
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The dorsal and ear tissues were formalin-fixed and paraffin-embedded, and stained with H&E. Epidermal thickness was accurately measured by ImagePro Plus software (Leeds Precision Instruments, Minneapolis, MN, USA). The total epidermal area was calculated using a series of rectangles and the data was divided by the total length of the epidermis.
For immunohistochemistry, sections from the ear and dorsal tissues were deparaffinized with xylene and rehydrated, and then hydrated with a graded alcohol series. The ear and dorsal sections were incubated in 10 ml citric acid (pH 6.0) at 95°C for 30 min to unmask antigens and the endogenous peroxidase activity was quenched by treating sections with 3% hydrogen peroxide at RT for 5 min. The sections were blocked at RT for 60 min followed by incubation with primary antibodies (Abs): rabbit anti-mouse IL-17A polyclonal Ab (H-132; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-mouse IL-22 polyclonal Ab (ab18564; Abcam, Cambridge, MA, USA) and rabbit anti-mouse IL-23 polyclonal Ab (H-113; Santa Cruz Biotechnology, Inc.). This was followed by treatment with horseradish peroxidase-linked secondary anti-rabbit GT Vision™ II polymer (Dako, Carpinteria, CA, USA) and DAB substrate kit for peroxidase (Dako).
For immunohistochemistry, sections from the ear and dorsal tissues were deparaffinized with xylene and rehydrated, and then hydrated with a graded alcohol series. The ear and dorsal sections were incubated in 10 ml citric acid (pH 6.0) at 95°C for 30 min to unmask antigens and the endogenous peroxidase activity was quenched by treating sections with 3% hydrogen peroxide at RT for 5 min. The sections were blocked at RT for 60 min followed by incubation with primary antibodies (Abs): rabbit anti-mouse IL-17A polyclonal Ab (H-132; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-mouse IL-22 polyclonal Ab (ab18564; Abcam, Cambridge, MA, USA) and rabbit anti-mouse IL-23 polyclonal Ab (H-113; Santa Cruz Biotechnology, Inc.). This was followed by treatment with horseradish peroxidase-linked secondary anti-rabbit GT Vision™ II polymer (Dako, Carpinteria, CA, USA) and DAB substrate kit for peroxidase (Dako).
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