The whole eyeballs were immersed in 4% paraformaldehyde for 24 h at room temperature. Subsequently, the eyeballs were treated with gradient alcohol and xylene and embedded in paraffin for sectioning at a thickness of 4 μm. Tissue sections stained with hematoxylin/eosin were used to observe the corneal structure. Immunohistochemical staining was performed according to our previously published protocol52 (link). Briefly, antigen retrieval was performed with sodium citrate repair solution by microwave heating, non-specific antigens were eliminated by 3% H2O2, and the sections were blocked with goat serum for 1 h and incubated with primary antibodies against IL-1β (1:100, ab254360, Abcam), TNF-α (1:1000, ab183218, Abcam), TRAF6 (1:300, ab33915, Abcam), IRAK1 (1:1000, ab238, Abcam), and COX2 (1:400, ab179800, Abcam) overnight at 4 °C. Next day, the sections were incubated with the Envision horseradish peroxidase system (Gene Tech, Shanghai, China) for 2 h at room temperature. The sections were finally incubated with 3,3′-diaminobenzidine (DAB) (Gene Tech, Shanghai, China) for 5 min. The staining was observed and captured with an Eclipse Ni microscope (Nikon, Tokyo, Japan).
3 3 diaminobenzidine (dab)
Manufactured by Gene Tech
Sourced in China
DAB is a laboratory equipment designed for the detection and visualization of specific biomolecules in various sample types. It functions as a chromogenic substrate that produces a brown-colored precipitate upon enzymatic reaction, allowing for the identification and localization of target analytes in histological or cytological preparations.
Automatically generated - may contain errors
Lab products found in correlation
Chemmate envision hrp, by Gene Tech (2 mentions)
Ab34710, by Abcam (2 mentions)
Ab5694, by Abcam (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Ab53707, by Abcam (2 mentions)
Hoechst 33258, by Solarbio (2 mentions)
Eclipse ni microscope, by Nikon (1 mentions)
Ab254360, by Abcam (1 mentions)
Ab33915, by Abcam (1 mentions)
Ab183218, by Abcam (1 mentions)
Ab179800, by Abcam (1 mentions)
Ab238, by Abcam (1 mentions)
Ab83760, by Abcam (1 mentions)
Light microscopy, by Sangon (1 mentions)
Hematoxylin, by Sangon (1 mentions)
Anti gp130, by Abcam (1 mentions)
Ab92824, by Abcam (1 mentions)
Igg2b isotype antibody, by Agilent Technologies (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Pbs t, by Thermo Fisher Scientific (1 mentions)
35 protocols using 3 3 diaminobenzidine (dab)
1
Immunohistochemical Analysis of Corneal Structure
2
Histological Analysis of Tibial Bone
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After fixation in 10% formaldehyde for 3 days, tibias were decalcified in 10% EDTA (PH7.4) for 21 days, dehydrated, and embedded in paraffin. Paraffin blocks contain tibias were sliced in a coronal for further TRAP staining and Hematoxylin-Eosin staining (H&E staining) and were analyzed under a high-quality microscope. The number of TRAP-positive multinucleated OCs, osteoclasts per bone surface (OCs/BS), and the percentage of BV/TV (%) were analyzed using ImagePro Plus 6.0 software. Sections (Tibias and Livers) were immunostained with anti-rabbit ALDH1A1 (1:200, proteintech) antibodies for 12h, followed by detection using a HRP-conjugated secondary antibody and 3,3′-Diaminoben- zidine (DAB) (Gene Tech, Shanghai, China) to measure the expression of proteins, and were analyzed under a high-quality microscope.
3
Immunohistochemical Staining of HGF Expression
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Immunohistochemical staining was performed by two-step procedure. Upon rehydration as above, the slides were subjected to antigen retrieval by pressure-cooking for 15 minutes. Endogenous peroxidase activity was neutralized using peroxide block placement on the slides for 10 minutes at room temperature. The slides were then incubated with anti-HGF polyclonal antibody (ab83760, Abcam, Cambridge, MA; diluted 1: 200) at 4°C overnight. This was followed by incubation with peroxidase-conjugated polymer (ChemMate EnVision / HRP; Gene Tech, Shanghai, China) for 30 minutes at room temperature. The chromogen reaction was developed in 3,3′-diaminobenzidine (DAB; Gene Tech, Shanghai, China) tetrahydrochloride for 5 minutes. Finally, hematoxylin was used as a light nuclear counterstain.
The percentage of positive-staining cells were graded on a scale of 0-3, with less than 5% positive-staining cells as grade 0, 5-25% as grade 1, 26-50% as grade 2, and more than 50% as grade 3. The intensity of staining also graded on a scale of 0-2, with negative to weak intensity as grade 0, weak-moderate intensity as grade 1, and moderate to strong intensity as grade 2. Finally, the score of percentage and intensity was multiplied. The final score between 0-2 was determined as low expression, and score higher than 2 was determined as high expression.
The percentage of positive-staining cells were graded on a scale of 0-3, with less than 5% positive-staining cells as grade 0, 5-25% as grade 1, 26-50% as grade 2, and more than 50% as grade 3. The intensity of staining also graded on a scale of 0-2, with negative to weak intensity as grade 0, weak-moderate intensity as grade 1, and moderate to strong intensity as grade 2. Finally, the score of percentage and intensity was multiplied. The final score between 0-2 was determined as low expression, and score higher than 2 was determined as high expression.
4
Immunohistochemical Staining of Tissue Sections
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The frozen tissue section slides were fixed in acetone for 15 minutes and subsequently washed by PBS. Endogenous peroxidases and non-specific binding were blocked by incubating with 3% hydrogen peroxide for 10 minutes and with 3% BSA, respectively. Slides were incubated with primary antibodies for 30 minutes at room temperature, followed by a 30-minute incubation with horseradish peroxidase-conjugated goat anti human IgG antibodies (Thermofisher). Antibody binding was visualized by incubating with the peroxidase substrate 3,3’-diaminobenzidine (DAB, GeneTech) for 5 minutes. After counterstaining with hematoxylin (Sangon Biotechnology), tissue sections were analyzed using light microscopy (Nikon).
5
Immunohistochemical Analysis of CPXM2 and gp130
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Briefly, following deparaffinization, rehydration and antigen retrieval, the slides were blocked with 5% normal bovine serum in TBST for 30 min at 37°C, and the slides were incubated with the anti-CPXM2 antibody (1:296 dilution; cat. no. ab201077, Abcam, at 1:100) and anti-gp130 (1:396 dilution; cat. no. 9145, Cell Signaling Technology, Inc) overnight at 4°C. Normal rat immunoglobulin G (1:196 dilution; cat. no. D110964, Sangon Biotech Co. Ltd.) was used instead of the aforementioned primary antibodies for the negative control. Then, after washing with PBS, the slides were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; cat. no. A0192, Beyotime Institute of Biotechnology) at RT for 1 h. Subsequently, the slides were visualized using 3,3-diaminobenzidine (DAB; Gene Tech, Shanghai, China) and counterstained using hematoxylin (H&E; Gene Tech, Shanghai, China). CPXM2 expression was semi-quantitatively characterized as high or low on account of a modified H score system as previously described.23 (link)
6
Immunohistochemical Staining of Human Cells in Nude Mice
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For immunohistochemical staining, samples were fixed with 4 % paraformaldehyde for 48 h at room temperature. Bone samples were decalcified for 3 weeks in 10 % EDTA (pH 7.4) with weekly changes, and then all the samples were embedded in paraffin. Anti-human mitochondria antibody (1:400; ab92824, Abcam, Cambridge, UK) were used for detecting human cells in nude mice. After dewaxed and rehydrated, sections(4μm) were blocked with 3 % hydrogen peroxide for 15–20 mins at room temperature, followed by incubation with pepsin solution digest-all (Invitrogen, USA) for 20 mins at room temperature for bone sections antigen retrieval. Other organs sections were placed in trisodium citrate buffer (pH = 6.0) for 40 mins at 95 °C. After naturally cooled to room temperature, the sections were washed with PBS for 3 times and incubated with the primary antibody solutions overnight at 4 °C. Sections were rinsed with deionized water and then PBS, followed by 30 mins incubation with horse reddish peroxidase working solution (Gene Tech, Shanghai) at room temperature for 30 mins. After washed, 50-100μl 3,3′-diaminobenzidine (DAB) (Gene Tech, Shanghai) working solution was used for 5–10 mins at room temperature. The sections were then lightly counterstained with hematoxylin.
7
Immunohistochemical Detection of p-Smad2
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Serial slides, each 3-um thick, were cut from paraffin-embedded tissue. One slide was stained with hematoxylin and eosin (HE). Immunohistochemical staining was performed on another slide, using the two-step procedure. The anti-human p-Smad2 rabbit polyclonal antibody (Ser465/467) (Chemicon, Billerica, MA, USA; diluted 1: 500) was used. After de-paraffinization and hydration, the slides were subjected to antigen retrieval by pressure-cooking for 30 minutes. Endogenous peroxidase activity was neutralized using peroxide block placement on the slides for 15 minutes at room temperature. The slides were then incubated with anti-p-Smad2 antibody for 40 minutes at 4°C. This was followed by incubation with peroxidase-conjugated polymer (ChemMate EnVision/HRP; Gene Tech, Shanghai, China) for 30 minutes at room temperature. The chromogen reaction was developed in 3,3′-diaminobenzidine (DAB; Gene Tech, Shanghai, China) tetrahydrochloride for 10 minutes. Finally, hematoxylin was used as a light nuclear counterstain. The negative control used was an IgG2b isotype antibody (Dako), ensuring the same concentration of immunoglobins as for anti-p-Smad2.
8
Immunohistochemical Analysis of CSTF2 in Liver Cancer
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The liver cancer tissues of mice were fixed with 4% PFA and embedded in paraffin. Approximately 5-μm thick slices were prepared for immunohistochemical staining. The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C. After being washed with PBS, the sections were incubated with biotinylated anti-rabbit Immunoglobulin G (IgG) secondary antibodies for 1 h at RT (1:200, Vector, Peterborough, United Kingdom). Then, the sections were visualized with a Vectastain ABC kit (Vector) at RT for 30 min and developed with 3,3’-diaminobenzidine (DAB) (Genetech, Shanghai, China) at RT. The images were visualized using a microscope. Tissue array chips were purchased from Servicebio (ZL-LivHCC961, Shanghai, China), and included HCC tissues (n = 48) and adjacent normal liver tissues (n = 48). Immunohistochemistry was executed on a tissue array chip using CSTF2 (1:200, Proteintech, China).
9
Immunohistochemical Analysis of TGF-β1, α-SMA, and Collagen I
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Sections were stained with primary antibodies against TGF‐β1 (ab92486, 1:200), α‐SMA (ab5694, 1:400) and Collagen I (ab34710, 1:400) (all obtained from Abcam), incubated at 4°C overnight, stained with secondary antibodies at room temperature for 30 minutes. Sections were then washed with PBS and incubated with 3,3'‐Diaminobenzidine (DAB) (Gene Tech, Shanghai, China) for 5 minutes at room temperature. The detailed procedure has been previously described.28
10
Immunohistochemical Analysis of Brain Tissue
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Paraffin-embedded brain tissue sections were deparaffinized in dimethylbenzene and rehydrated in ethanol. The activity of endogenous peroxidase was inactivated by incubating in 3% H2O2 at 37 °C for 10 min. The sections were immersed in citrate buffer (pH = 6.0), heated to 100 °C and cooled at room temperature for antigen recovery. After nonspecific binding sites were blocked in 5% goat serum containing Triton X-100 (0.2% v/v) for 2 h, the sections were then combined with GluR1 (1:200, Santa Cruz, USA), Iba1 (1:1000, Abcam, UK), and SYN (1:200, Abcam, UK) antibodies in IHC antibody diluent overnight at 4 °C. After washing in phosphate-buffered saline (PBS), horseradish peroxidase-conjugated secondary anti-rabbit antibodies (Gene Tech, USA) were used for 2 h. The reaction was developed with a 3,3′-diaminobenzidine (DAB, Gene Tech, USA) kit and counterstained with haematoxylin. Finally, images were taken on a microscope (Olympus, Tokyo, Japan) at a magnification of 200 ×, and the averaged optical densities (AOD) of the target protein as well as the number of Iba1-labelled microglia were calculated with ImageJ. The data were analysed by GraphPad Prism 5.0.
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