The paraffin-embedded skin sections were deparaffinized, rehydrated, and blocked with 3% hydrogen peroxide and then incubated at 37°C for 1 h with anti-CD19 antibody (1:1,600; Cell Signaling Technology, Inc., Danvers, MA, USA). Thereafter, the sections were washed with phosphate-buffered saline (PBS) and incubated at room temperature for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:200; Abcam). The reaction was subsequently visualized with 3,3′-diaminobenzidine solution (Gene Tech Co., Ltd., Shanghai, China), and the sections were counterstained with hematoxylin. Semi-quantification of the IHC staining results was performed under a high-power field (400×).
3 3 diaminobenzidine solution
Manufactured by Gene Tech
Sourced in China
3,3′-diaminobenzidine solution is a chemical compound commonly used in histochemical and immunohistochemical procedures. It serves as a chromogenic substrate for enzymatic reactions, resulting in the production of a colored precipitate that can be visualized under a microscope.
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Lab products found in correlation
3 protocols using 3 3 diaminobenzidine solution
1
Immunohistochemical Analysis of CD19 in Skin
2
Immunohistochemical Analysis of MARCO Expression
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After deparaffinization with dimethylbenzene, tissue sections were sequentially rehydrated using graded alcohol. Antigen retrieval was achieved by boiling sections in citrate–disodium hydrogen phosphate buffer (pH 6.0) under high pressure for 5 min. Endogenous peroxidase was inactivated by incubating with 0.3% hydrogen peroxide for 30 min. Subsequently, slides were overnight incubated with primary antibodies (diluted 1:100) against MARCO (Abcam, MA, USA) at 4°C. Sections were incubated in biotinylated secondary antibody and streptavidin peroxidase (Invitrogen, NE, USA) and then developed with 3,3′-diaminobenzidine solution (Genetech, Shanghai, China) before counter-staining with hematoxylin. The intensity and extent of MARCO immunostaining were evaluated for all samples under double-blinded conditions. The percentage of positive staining was scored as 0 (0–9%), 1 (10–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%), with the intensity scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (dark staining). The total score was calculated as the product of intensity and extent, ranging from 0 to 12. A total score of ˂6 was defined as low MARCO expression, while ≥6 was defined as high MARCO expression. Two pathologists independently assessed the specimens. Images were obtained using an Olympus BX63 microscope (Olympus, Tokyo, Japan).
3
FAT10 Immunohistochemistry Protocol
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After deparaffinization with dimethylbenzene, tissue sections were sequentially rehydrated with graded alcohols. The antigens were retrieved by boiling the sections in citrate–disodium hydrogen phosphate buffer (pH 6.0) with high pressure for 5 minutes. Endogenous peroxidase was inactivated by incubation with 0.3% hydrogen peroxide for 30 minutes. The slides were subsequently incubated with the primary antibody against FAT10 (ab134077, Abcam, USA) overnight at 4°C, incubated with the biotinylated secondary antibody and streptavidin peroxidase (Invitrogen, Grand Island, USA) for 30 minutes at 37°C, and developed with 3,3′-diaminobenzidine solution (GeneTech, Shanghai, China) before counter-staining with hematoxylin.
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