Biocytin Fills: To reliably reconstruct the fine axonal branches of cortical neurons, dedicated experiments were performed following the classical avidin-biotin-peroxidase method. Biocytin (Sigma) was added to the intracellular solution at a high concentration (5–10 mg/ml), which required extensive sonication. At the end of recordings, the patch pipette was removed carefully until obtaining an inside out patch. The slice was then left in the recording chamber for at least further 5–10 min to allow further diffusion. Slices were then fixed with 4% paraformaldehyde in phosphate buffer saline (PBS, Sigma) for at least 48 hr. Following fixation, slices were incubated with the avidin-biotin complex (Vector Labs) and a high concentration of detergent (Triton-X100, 5%) for at least two days before staining with 3,3′Diaminobenzidine (DAB, AbCam). Cells were then reconstructed and cortical layers delimited using Neurolucida 7 (MBF Bioscience) and the most up to date mouse atlas (Allen Institute).
3 3 diaminobenzidine dab
Manufactured by Abcam
Sourced in United Kingdom, United States
3,3′-Diaminobenzidine (DAB) is a chromogenic substrate used in immunohistochemistry and immunocytochemistry applications to visualize target antigens. It produces a brown color reaction when oxidized by peroxidase enzymes, making it a commonly used agent for the detection and localization of proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Biocytin, by Merck Group (2 mentions)
Neurolucida 7, by MBF Biosciences (2 mentions)
Avidin biotin complex, by Vector Laboratories (2 mentions)
Fgf2 sc 74412, by Santa Cruz Biotechnology (1 mentions)
Anti vegf a sc 7269, by Santa Cruz Biotechnology (1 mentions)
Anti ki67, by Santa Cruz Biotechnology (1 mentions)
Ab13840, by Abcam (1 mentions)
Vectastain elite abc rabbit igg kit, by Vector Laboratories (1 mentions)
Hydrogen peroxide, by Abcam (1 mentions)
Rankl, by Bioss Antibodies (1 mentions)
Interleukin 6 (il 6), by Bioss Antibodies (1 mentions)
Il 1β, by Bioss Antibodies (1 mentions)
Abc detection kit, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Entellan, by Merck Group (1 mentions)
Sirt1, by Bioss Antibodies (1 mentions)
Mayer s hematoxylin, by Thermo Fisher Scientific (1 mentions)
Modified russell movat pentachrome, by Abcam (1 mentions)
12 protocols using 3 3 diaminobenzidine dab
1
Detailed Biocytin Labeling Protocol
2
Immunohistochemical Evaluation of Angiogenic Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immunohistochemistry staining was used to evaluate VEGFA and FGF-2 positive expressions in the tissues. The histopathology slides were processed immunohistochemically using horse radish-labeled monoclonal antibodies (anti-VEGFA #SC-7269 (
Santa Cruz BiotechnologyInc., California, USA), FGF-2 #SC-74412 (
Santa Cruz BiotechnologyInc., California, USA)), and 3–3′ diaminobenzidine (DAB) (Abcam, USA). Then, the positive expression of the protein marked by brown precipitate on the cells of the oral ulcer site was observed using an inverted light microscope with 100 × , 400 × , and 1000× magnification in five different fields of view by two observers.
Santa Cruz BiotechnologyInc., California, USA), FGF-2 #SC-74412 (
Santa Cruz BiotechnologyInc., California, USA)), and 3–3′ diaminobenzidine (DAB) (Abcam, USA). Then, the positive expression of the protein marked by brown precipitate on the cells of the oral ulcer site was observed using an inverted light microscope with 100 × , 400 × , and 1000× magnification in five different fields of view by two observers.
3
Immunohistochemical Analysis of Chagas Disease
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed a descriptive immunohistochemistry analysis of samples from patients in the CCC group with the aim of generating hypotheses about the expression of different molecules potentially important for the pathophysiology of Chagas disease. For this purpose, tissue sections were collected on poly-L-lysine coated slides and dried in a convection oven at 90 °C for 24 h. Then, the classical protocol for sections was followed: deparaffinization followed by hydration. To unmask the antigen, the slides were boiled in a sodium citrate solution at pH 6 for 1 h. After boiling and cooling, the slides were washed in distilled water for 15 min. Endogenous peroxidase blocking was performed by incubating the slides in 3% hydrogen peroxide for 30 min at room temperature, followed by washing in distilled water for 10 min and washing in 1% phosphate-buffered saline (PBS) solution for five minutes. Nonspecific sites were then blocked using 2% skim milk for 30 min. The sections were then incubated with antibodies against type IV collagen (ab 34710) and XIVA1 (NBP1-86877) using a 1:50 dilution for 18 h (overnight) in a refrigerator at 4 °C. Secondary detection was performed with a peroxidase-conjugated goat anti-rabbit secondary antibody at 1:10,000 for 45 min at room temperature. The signal was detected using 3,3′ Diaminobenzidine (DAB) (Abcam), and the reaction was quenched in 1% PBS.
4
Immunohistochemical Analysis of Matrix Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Matrix protein expression (collagen I and fibronectin) was analyzed using immunohistochemical staining methods. For antibody incubation, 3µm thick paraffin sections were mounted on poly-L-lysine coated glass slides. Polyclonal rabbit antibodies against collagen type I and fibronectin were used. Detection of the bound antibodies was performed using a HRP second antibody and using 3, 3-diaminobenzidine (DAB) as selenium organic reagent, according to the manufacturer's instructions (Abcam, England). Control experiments were performed omitting the first antibody and using phosphate-buffered saline instead. The integrated optical density of the positive material in each glomerulus and the glomerular area were measured by the morphological analysis system, the ratio of which showed the relative content of FN and Col I in the renal glomeruli.
5
Immunohistochemical Analysis of Ovarian Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ovarian paraffin sections were dewaxed with xylene and rehydrated with an alcohol gradient before IHC analysis. After heating and repair in sodium citrate solution, the samples were blocked with H2O2 and 10% horse serum. Sections were incubated at 4 °C overnight with primary antibodies, including anti-CLEAVED CASPASE3 (1:400, CST #9664, USA), anti-KI67 (1:500, Santa Cruz #sc-15402, USA), anti-ILK1(1:100, CST #3862, USA), and anti-MVH (1:200, Abcam #ab13840, UK). The next day, all sections were washed with PBS and incubated with secondary antibodies for 1 h at room temperature. Bound antibodies were detected using a biotin-streptavidin horseradish peroxidase(HRP) detection system (ZSGB-BIO #SP-9000, China). 3,3’-diaminobenzidine (DAB) (Abcam, UK) staining was performed for 2 min at room temperature. Cell nuclei were counterstained with Mayer’s hematoxylin for 5 min.
Five fields in each section were randomly selected for examination. Immunoreactive scores (IRS) (Remmele & Stegner, 1987 ) were used to score the staining results. Briefly, the intensity of the staining signal was classified as “0” (negative), “1” (weak), “2” (moderate), or “3” (strong) and the extent of the stained cells was classified as “0 (<5%), “1 (5%–25%), “2” (25%–50%), “3” (50%–75%), or “4” (>75%). The final IHC staining score was obtained by multiplying the two fractions together.
Five fields in each section were randomly selected for examination. Immunoreactive scores (IRS) (Remmele & Stegner, 1987 ) were used to score the staining results. Briefly, the intensity of the staining signal was classified as “0” (negative), “1” (weak), “2” (moderate), or “3” (strong) and the extent of the stained cells was classified as “0 (<5%), “1 (5%–25%), “2” (25%–50%), “3” (50%–75%), or “4” (>75%). The final IHC staining score was obtained by multiplying the two fractions together.
6
Immunohistochemical Analysis of Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sections were stained with the following primary antibodies for the IHC analyses: IL-1β, IL-6, RANKL, and Cathepsin K (Bioss, Woburn, MA, USA) (1:400 dilution). After deparaffinization and rehydration, sections were treated with 3% hydrogen peroxide (Abcam) for 10 min to quench the endogenous peroxidase activity, followed by the incubation with normal goat serum to block the non-specific binding for 30 min at room temperature. Primary antibodies with different specific concentrations were applied to the sections and incubated overnight at 4 °C. The following day, the slides were incubated with the biotinylated secondary antibody for 30 min using the VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Labs, Burlingame, CA, USA). Subsequently, the prepared VECTASTAIN ABC reagent was applied to the slides and incubated for another 30 min. Sections were stained with 3,3’-Diaminobenzidine (DAB) (Abcam) and counterstained with hematoxylin.
7
Immunohistochemical Analysis of Tumor CD31
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All harvested tumors were fixed with 4% paraformaldehyde overnight in a cold room and then embedded with paraffin. On the day of immunohistochemistry, the paraffin-embedded tumor tissues were deparaffinized and rehydrated. Sections were pretreated with heat-mediated antigen retrieval using Tris/EDTA pH 9.0 buffer. Then, sections were incubated with hydrogen peroxidase blocking agent (Abcam) for 20 minutes at room temperature and washed with phosphate-buffered saline twice for 3 minutes each time. According to the manufacturer’s protocol for ABC detection kit (Abcam), 3,3′-diaminobenzidine (DAB) staining was performed to detect CD31 (Abcam; 1:200 overnight incubation at 4°C).
8
Immunohistochemical Analysis of HIF-1α
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain tissue sections (n = 6)were dewaxed, dehydrated, and blocked step by step with 3% hydrogen peroxide-formalin. After a drop-wise addition of 50 μl goat sera (Santa Cruz, USA), the sections were incubated with 50–100 μl primary HIF-1α antibody (Santa Cruz, USA) for 10 min at 37°C and hydro-incubated for 2 h at 37°C. After rinsing thrice with 50 μl 0.1 mol/L PBS, a reinforcing agent was added and hydro-incubated for 30 min. The sections were labeled with freshly prepared 3,3’-diaminobenzidine (DAB) (Abcam, UK), counter-stained with hematoxylin, dehydrated, and mounted with neutral gum. Brownish yellow stain was positive for HIF-1α expression. Moreover, HIF-1α- positive cells were measured from six visual fields with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, Maryland, USA).
9
Immunohistochemical Evaluation of Astrocyte and Microglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Astrocyte glial fibrillary acidic protein (GFAP) and microglia (CD11) were counted in order to evaluate astrocyte and microglial activity. The sections were immunostained using the polyclonal primary antibody for GFAP (1:400) (PA1239) or CD11 (1:500) (DB Biotech).
The sections were deparaffinized in xylene, hydrated through a graded ethanol series, and washed in running water. After antigen retrieval, the sections were incubated with rabbit primary antibody GFAP [25] (link) and mouse primary antibody CD11 [26] (link) overnight at 4 °C. Afterwards, the sections were rinsed for 10 min with phosphate buffer solution (PBS) and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies (Santa Cruz Biotechnology, Inc., USA) at the dilution of 1:200 for 1 h at room temperature. After washing in PBS, peroxidase activity was detected with 3, 3-diaminobenzidine (DAB, Abcam, UK) as the chromogenic substrate. The sections were counterstained with he-matoxylin (Sigma-Aldrich, USA), dehydrated in an increasing alcohol series, cleared in xylene, and finally mounted on Entellan® (Merck, Germany). Astrocytes showing GFAP and CD11+ cells were manually counted in three microscopic fields (0.107 mm2; 89.82 × 120.70 µm) of the immunohistochemically stained sections from the cerebellum and hippocampus. Results are expressed as the average number of cells/0.1 mm2.
The sections were deparaffinized in xylene, hydrated through a graded ethanol series, and washed in running water. After antigen retrieval, the sections were incubated with rabbit primary antibody GFAP [25] (link) and mouse primary antibody CD11 [26] (link) overnight at 4 °C. Afterwards, the sections were rinsed for 10 min with phosphate buffer solution (PBS) and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies (Santa Cruz Biotechnology, Inc., USA) at the dilution of 1:200 for 1 h at room temperature. After washing in PBS, peroxidase activity was detected with 3, 3-diaminobenzidine (DAB, Abcam, UK) as the chromogenic substrate. The sections were counterstained with he-matoxylin (Sigma-Aldrich, USA), dehydrated in an increasing alcohol series, cleared in xylene, and finally mounted on Entellan® (Merck, Germany). Astrocytes showing GFAP and CD11+ cells were manually counted in three microscopic fields (0.107 mm2; 89.82 × 120.70 µm) of the immunohistochemically stained sections from the cerebellum and hippocampus. Results are expressed as the average number of cells/0.1 mm2.
10
Immunohistochemical Analysis of SIRT1, ET-1, and Aquaporins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein expression of SIRT1 (Bioss Antibodies, USA, 1:200), ET-1 (Bioss Antibodies, USA, 1:200), AQP1 (Scervicebio Co., USA, 1:1000), AQP4 (Scervicebio Co., USA, 1:1500), AQP5 (ABclonal, USA, 1:200) and autophagy biomarkers LC3 (Abcam, USA, 1:1200), P62 (ABclonal, USA, 1:200) were determined in each group by incubating tissue sections in primary antibodies overnight followed by incubation with secondary antibodies to perform IHC. The visualization of slides was detected using 3,3-Diaminobenzidine (DAB, Abcam, USA), and counterstained with hematoxylin. Then, the sections were analyzed and photographed using an Olympus microscope (Japan) with installed camera. The positive reaction was thresholded and calculated in relation to the surface area using Image J. The data were then decoded and statistically analyzed.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!