All IHC staining was performed on formalin-fixed and paraffin-embedded tissue sections, 4μm in thickness. Sections were deparaffinized, rehydrated and endogenous peroxidase activity blocked by incubation with 3% hydrogen peroxide in 100% alcohol (1:1) for 15 min. Antigen retrieval (for CXCL1) was performed using target retrieval solution (pH 6.0; Dako) and heat induction (120°C at 15psi) for 30 sec. Sections were incubated with following primary antibodies: rabbit anti-CXCL1 (Protein Tech, 1:100), goat anti-DMPK (Santa Cruz, 1:25), and rabbit anti-Neurabin 1 (Abcam, 1:500). Exposure to primary antibodies was followed by incubation with appropriate HRP-labeled polymer secondary antibodies (Dako) and staining visualized with a 3,3′-diaminobenzidine (DAB)/substrate-chromogen system (Dako). Gill’s hematoxylin was used for counterstaining. For anti-DMPK staining sections were treated separately with avidin, biotin and 3% rabbit serum for 10 min followed by primary antibody incubation and subsequent exposure to biotinylated rabbit anti-goat secondary antibody/streptavidin-HRP for 30 min. Control tissues included macroscopically normal renal cortex taken from kidneys resected for localized neoplasia, brain, skin, intestine, heart, tonsil, skeletal muscle, and ovarian carcinoma. Semiquantitative scoring of stained kidney sections was performed in a blinded fashion.
3 3 diaminobenzidine dab substrate chromogen system
Manufactured by Agilent Technologies
Sourced in United States
The 3,3′-diaminobenzidine (DAB)/substrate-chromogen system is a laboratory reagent used for colorimetric detection in various applications, such as immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). It functions as a chromogenic substrate that produces a brown precipitate upon catalytic reaction, enabling the visual identification and localization of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti cxcl1, by Proteintech (2 mentions)
Goat anti dmpk, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti neurabin 1, by Abcam (2 mentions)
Hrp labeled polymer secondary antibodies, by Agilent Technologies (2 mentions)
Baf1097, by R&D Systems (1 mentions)
Entellan, by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
4 protocols using 3 3 diaminobenzidine dab substrate chromogen system
1
Immunohistochemical Analysis of Kidney Tissue
2
Immunohistochemical Analysis of Kidney Tissue
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All IHC staining was performed on formalin-fixed and paraffin-embedded tissue sections, 4μm in thickness. Sections were deparaffinized, rehydrated and endogenous peroxidase activity blocked by incubation with 3% hydrogen peroxide in 100% alcohol (1:1) for 15 min. Antigen retrieval (for CXCL1) was performed using target retrieval solution (pH 6.0; Dako) and heat induction (120°C at 15psi) for 30 sec. Sections were incubated with following primary antibodies: rabbit anti-CXCL1 (Protein Tech, 1:100), goat anti-DMPK (Santa Cruz, 1:25), and rabbit anti-Neurabin 1 (Abcam, 1:500). Exposure to primary antibodies was followed by incubation with appropriate HRP-labeled polymer secondary antibodies (Dako) and staining visualized with a 3,3′-diaminobenzidine (DAB)/substrate-chromogen system (Dako). Gill’s hematoxylin was used for counterstaining. For anti-DMPK staining sections were treated separately with avidin, biotin and 3% rabbit serum for 10 min followed by primary antibody incubation and subsequent exposure to biotinylated rabbit anti-goat secondary antibody/streptavidin-HRP for 30 min. Control tissues included macroscopically normal renal cortex taken from kidneys resected for localized neoplasia, brain, skin, intestine, heart, tonsil, skeletal muscle, and ovarian carcinoma. Semiquantitative scoring of stained kidney sections was performed in a blinded fashion.
3
Immunohistochemical Staining of Endoglin
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Immunohistochemistry was performed as described before (23 (link)). In short, FFPE sections (4 μm) were deparaffinized, blocked in 0.3% hydrogen peroxide (H2O2) in methanol for 20 min, and rehydrated. Antigen retrieval was performed by boiling the sections in a 0.01 M citrate solution (pH 6.0) for 10 min. After being washed with phosphate-buffered saline (PBS), the sections were incubated with polyclonal goat anti-human endoglin (1:400—BAF1097, R&D systems, MN, United States) in PBS/1% bovine serum albumin (BSA), and left overnight at room temperature. The next day, the slides were washed and then incubated with a biotinylated polyclonal rabbit anti goat secondary antibody (Dako, CA, United States) for 30 min, washed and incubated with Vectastain complex (Vector Laboratories, CA, United States). The color was developed using a 3,3’diaminobenzidine (DAB) + substrate chromogen system (Dako, CA, United States), following the manufacturer instructions. Nuclear staining was performed using hematoxylin (Merck, Darmstadt, Germany). Slides were dehydrated and mounted using entellan (Merck, Darmstadt, Germany).
4
Histological Analysis of Mouse Kidneys
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Mice kidneys were fixed in 4% paraformaldehyde for paraffin-embedded kidney sections (3 μm), which were then deparaffinized and rehydrated for the following staining techniques. For kidney histologic examination, periodic acid–Schiff (PAS) staining was performed using the standard methods. For immunohistochemistry, 3% H2O2 was used to remove endogenous peroxidase, and the antigen was retrieved in Tris-EDTA (TE) buffer, following by blocking with 5% normal goat serum and incubating with the primary antibody at 4°C overnight (WT-1: 1 : 100). After washing three times in PBS, the sections were then incubated with the appropriate secondary antibodies for 1 h in room temperature, washed with PBS three times again, and the signals were visualized using liquid 3,3'-Diaminobenzidine (DAB) + substrate chromogen system (Dako, USA), followed by counterstaining with hematoxylin and capturing images using a multiple viewing microscope (Nikon).
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