Immunohistochemistry staining of formalin-fixed and paraffin-embedded tissues was performed as previously described26 (link). After micro-sectioning at 4 μm thickness, the sections underwent heat-induced antigen retrieval for 3 min in EDTA buffer (pH 9.0; Dako, Carpinteria, CA). Sectioned slides were then incubated overnight with anti-human NIS (1:100 dilution; Santa Cruz Biotechnology, #sc-134515) at 4 °C. This was followed by incubation with HRP-labeled polymer-conjugated secondary antibodies against rabbit IgG (Dako) for 30 min at room temperature. A color reaction was induced using a ready-to-use 3,3′-diaminobenzidine substrate-chromogen solution (Dako) for 5 min, followed by washing with distilled water. Finally, sections were lightly counterstained with Mayer’s hematoxylin for 30 s before dehydration and mounting.
3 3 diaminobenzidine substrate chromogen solution
Manufactured by Agilent Technologies
Sourced in United States
3,3′-diaminobenzidine substrate-chromogen solution is a laboratory reagent used in various immunohistochemical and enzyme-based assays. It functions as a chromogenic substrate that undergoes a color change reaction upon enzymatic catalysis, enabling visualization and detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Hrp labeled polymer conjugated secondary antibodies against rabbit igg, by Agilent Technologies (1 mentions)
Edta buffer, by Agilent Technologies (1 mentions)
Podocin, by Santa Cruz Biotechnology (1 mentions)
Nephrin, by Progen Biotechnik (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Kim 1, by Santa Cruz Biotechnology (1 mentions)
Hsp70, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Hsp70, by Enzo Life Sciences (1 mentions)
Hmgb1, by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Kim 1, by R&D Systems (1 mentions)
Anti stra6 antibody, by Abcepta (1 mentions)
Anti collagen 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Eclipse te2000 s microscope, by Nikon (1 mentions)
4 protocols using 3 3 diaminobenzidine substrate chromogen solution
1
Immunohistochemical Staining of NIS in FFPE Tissues
2
Immunostaining and Immunoblotting for Kidney Injury
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For the immunohistochemical staining, paraffin-embedded sections (5 µm thick) were incubated with primary antibodies against mouse TLR2 (Abcam), TLR4 and cubilin (Santa Cruz), Kim-1 (R&D) and cleaved caspase 3 (Cell Signaling); or human TLR2 and TLR4 (Santa Cruz), HSP70 (Enzo Life Science) and HMGB1 (Abcam). Slides were developed using 3,3′-diaminobenzidine substrate-chromogen solution (Dako). For the immunofluorescence staining, frozen sections (12 µm thick) were incubated with antibodies against HSP60 and HSP70 (Enzo Life Science), and biglycan and HMGB1 (Abcam) followed by secondary antibodies conjugated with Alexa Fluor dyes (Invitrogen). For the immunobloting, proteins were probed with antibodies against Kim-1, podocin, HSP70 (Santa Cruz), HMGB1 (Abcam), nephrin (PROGEN), caspase 3 (Cell Signaling), β-actin and α-tubulin (Sigma-Aldrich).
3
Kidney Immunohistochemistry Analysis
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After deparaffinization and rehydration, kidney sections of saline-, L1-, or L5-injected mice, or L5-injected LOX1−/− mice were placed in 0.01 M sodium citrate buffer (pH 6.0) and heated in a microwave oven for 2.5 min at 720 W. For Mason’s trichrome stain, sections were stained according to the protocol of the manufacturer (Sigma-Aldrich). For IHC, sections were washed in PBS and incubated with 1% BSA for 30 min to block nonspecific staining. Sections were drained and incubated for 3 h at room temperature in a humidity chamber with respective antibody for IHC, including anti-STRA6 antibody (ABGENT), anti-collagen 1 antibody (Santa Cruz Biotechnology Inc.), or anti-vitamin A antibody (MyBioSource, San Diego, CA) diluted with antibody diluent (Dako, Carpentaria, CA). After washing in PBS, endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in methanol for 20 min, followed by sequential 10 min incubations with biotinylated link antibody and peroxidase-labeled streptavidin (Dako). Staining was completed after incubation with 3,3’-diaminobenzidine substrate-chromogen solution (Dako), and then counterstained with hematoxylin. Images from similar regions of sections of kidney were captured by bright field microscopy at 400× microscopic magnification.
4
Immunohistochemical Staining Protocol for FBXO32 and SMAD4
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FBXO32 and SMAD4 were stained with the two-step staining method of EnVision™, and the major steps are described as follows. Tissue sections were dewaxed in xylene, rehydrated in alcohol, and immersed in 3% hydrogen peroxide for 10 min to suppress endogenous peroxidase activity. Antigen retrieval was conducted by heating each section at 100°C for 30 min in 0.01 mol/l sodium citrate buffer (pH 6.0). After three 5-min rinses in phosphate-buffered saline (PBS), the sections were incubated for 1 h at room temperature with a mouse polyclonal anti-FBXO32 antibody (Epitomics, UK) diluted 1: 50 in PBS, and the bound antibodies were detected with a streptavidin-biotin-peroxidase system (Dako, USA) and 3,3′-diaminobenzidine substrate-chromogen solution (Dako, USA). The slides were counterstained with hematoxylin and inspected by an experienced pathologist. The staining area was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%) based on the percentage of positively stained cells [13 (link)]. The images were captured with an inverted Nikon Eclipse TE2000-S microscope (Tokyo, Japan) with 200× magnification, and then, the presence of green fluorescence indicative of a-SMA was qualitatively analyzed by 2 investigators blinded to the study. The degree and intensity of staining were independently assessed by 2 pathologists in a blinded manner.
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