To stain CD14+ monocytes, iPS-MLs and SH-SY5Y cells were cultured with the above-described TTRs, and re-suspended at a density of 2 × 105 cells/mL in DMEM medium (Gibco). Next, cells were adhered to slide glass using Cytospin, and fixed in 4% formalin phosphate buffer. After proteolytic activation for 10 min by proteinase K (Dako), samples were treated with methanol containing 0.3% H2O2 for 20 min to remove endogenous peroxidase activity. PBS containing 5% skimmed milk was used to block nonspecific background staining. Polyclonal rabbit anti-human TTR antibody (Dako) was diluted (1:100) in Dako REAL Antibody Diluent (Dako), and used as the primary antibody. Envision System-HRP Labelled Polymer Anti-Rabbit (Dako) was used as the secondary antibody. The reaction was visualized using the Dako Envision + kit/HRP (DAB) (Dako). TTR+ cells in CD14+ monocytes, iPS-MLs and SH-SY5Y cells were counted. Briefly, five visual fields were randomly chosen from each stained section, and the TTR+ cell number in each visual field determined. The average number of TTR+ cells per visual field was calculated.
Dako envision kit hrp dab
Manufactured by Agilent Technologies
Sourced in Japan
The Dako Envision + kit/HRP (DAB) is a lab equipment product designed for immunohistochemistry (IHC) applications. It is a polymer-based detection system that utilizes horseradish peroxidase (HRP) enzyme and 3,3'-diaminobenzidine (DAB) as the chromogenic substrate.
Automatically generated - may contain errors
Lab products found in correlation
Dako real antibody diluent, by Agilent Technologies (1 mentions)
Envision system hrp labelled polymer anti rabbit, by Agilent Technologies (1 mentions)
Polyclonal rabbit anti human ttr antibody, by Agilent Technologies (1 mentions)
Proteinase k, by Agilent Technologies (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Ba 1400, by Vector Laboratories (1 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Can get signal immunostain solution a, by Toyobo (1 mentions)
Vectastain abc reagent, by Vector Laboratories (1 mentions)
Biotinylated anti rabbit igg, by Vector Laboratories (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
5 protocols using dako envision kit hrp dab
1
Staining of CD14+ Monocytes, iPS-MLs, and SH-SY5Y Cells
2
Tissue Immunohistochemistry for DEC1 and pAMPK
3
Quantifying Apoptotic Osteocytes via Caspase-3 IHC
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Immunohistochemistry for caspase-3 was performed to detect apoptotic osteocytes. Deparaffinized sections of the knee were incubated with methanol containing 3% H2O2 for 30 min, followed by incubation with 1% normal horse serum in 0.01 M phosphate-buffered saline (pH 7.4) for 30 min. After sections were incubated with an anti-caspase-3 antibody (No. GTX110543, 1:4000 dilution; GeneTex, California, LA, USA) overnight at 4°C, a secondary antibody (horse biotinylated anti-mouse/rabbit IgG, 1:250 dilution; BA-1400, Vector Laboratories, Burlingame, CA, USA) was applied for 30 min at room temperature. The negative control was incubated with vehicle (1% bovine serum albumin), instead of an anti-caspase-3 antibody. The sections were then incubated with a streptavidin-biotin complex (1:50 dilution; Elite ABC, Vector Laboratories, Burlingame, CA, USA) for 30 min, and the immunoreaction was visualized using Dako EnVision + kit/HRP (DAB) (Dako Japan, Tokyo, Japan). Finally, counterstaining with hematoxylin was performed to stain nuclei. Six fields of view located between the articular cartilage and the growth plate (similar regions to those used to assess marrow adiposity and trabecular bone area) were photographed at 40× magnification. Total and caspase-3 positive osteocytes were counted, and the percentage of caspase-3 positive osteocytes was calculated.
4
Histological Analysis of Aneurysm Specimens
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After aneurysm resection, specimens were gently washed out with saline to remove any blood and then fixed in 10% formalin. For classical histological stainings, sections were stained with hematoxykin-eosin (HE) and Elastica-Masson (EM) methods. We also performed phospho tungstic acid hematoxylin (PTAH) stainings to detect and confirm fibrin components of the specimens.
PTAH staining was originally established by Mallory for glial fiber staining, but now it is also used for the demonstration of fibrin [8, 12, 22, 23, 26] . Although the mechanism of the staining reaction obtained by PTAH is largely unknown, PTAH stains the collagen pink, glial fibers, muscle fibers, and fibrin blue [22, 23, 26] . As for immunohistochemistry, we used CD68 as a marker of macrophage infiltration. The deparaffinized sections were processed through antigen retrieve for 15 min, and then were incubated with primary mouse monoclonal antibody against human CD68 (Dako, 1:100). Then, the immunoreactivity was visualized by treating with Dako Envision kit HRP (DAB) (Dako). Finally, sections were counterstained with hematoxylin.
PTAH staining was originally established by Mallory for glial fiber staining, but now it is also used for the demonstration of fibrin [8, 12, 22, 23, 26] . Although the mechanism of the staining reaction obtained by PTAH is largely unknown, PTAH stains the collagen pink, glial fibers, muscle fibers, and fibrin blue [22, 23, 26] . As for immunohistochemistry, we used CD68 as a marker of macrophage infiltration. The deparaffinized sections were processed through antigen retrieve for 15 min, and then were incubated with primary mouse monoclonal antibody against human CD68 (Dako, 1:100). Then, the immunoreactivity was visualized by treating with Dako Envision kit HRP (DAB) (Dako). Finally, sections were counterstained with hematoxylin.
5
Immunohistochemical Analysis of Ror2 Expression
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The sections were permeabilized with 0.1% (v/v) Triton X-100 for 20 min, washed in PBS, and then immersed in 0.1% (v/v) hydrogen peroxide for 60 min. After washing with PBS and blocking with Blocking serum (VECTASTAIN ABC KIT; Vector Laboratories, Burlingame, CA, USA) for 60 min, the specimens were incu-bated with anti-Ror2 rabbit polyclonal antibody (Kani et al., 2004) diluted 1:100 in Can Get Signal immunostain Solution A (Toyobo, Osaka, Japan) overnight at 4°C, followed by incubation with biotinylated anti-rabbit IgG (Vector Laboratories) for 120 min at room temperature. The sections were incubated with VECTASTAIN ABC reagent (Vector Laboratories) for 60 min at room temperature, developed with DAKO ENVISION kit/HRP (DAB) (DAKO, Carpinteria, CA, USA), and counterstained with hematoxylin. The slides were evaluated under the light microscope (BZ-X710).
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