Procedures for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting of plasma adipsin are described in Iizuka et al. (2010) [20 (link)]. In brief, centrifuged plasma samples (n = 2) were mixed with SDS-PAGE sample buffer containing 100 mM dithiothreitol. After separation through an SDS-PAGE gel (12% w/v acrylamide), protein bands were transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA), followed by incubation with anti-adipsin antibody (M-120, sc-50419, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubating the membrane with a secondary antibody against rabbit (Santa Cruz Biotechnology), adipsin was visualized by reaction with streptavidin-peroxidase and then with 20% (w/v) 3,3′-diaminobenzidine tetrahydrochloride (Nacalai Tesque, Kyoto, Japan) in 50 mM Tris-HCl, pH 7.6, 0.1% (v/v) H2O2 for 1 h. The membrane was then washed three times with distilled water.
3 3 diaminobenzidine tetrahydrochloride
Manufactured by Nacalai Tesque
Sourced in Japan
3,3′-diaminobenzidine tetrahydrochloride is a chemical compound commonly used as a chromogenic substrate in various laboratory applications. It is a crystalline solid that is soluble in water and other polar solvents.
Automatically generated - may contain errors
3 protocols using 3 3 diaminobenzidine tetrahydrochloride
1
Plasma Adipsin Immunoblotting Protocol
2
Histochemical Analysis of Colon Tissue
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Colon tissue samples were fixed in 10% formalin, embedded in paraffin, deparaffinized,
and retrieved as described previously [4 (link)]. The colon
sections were immunostained with various antibodies by using second antibodies conjugated
with horseradish peroxidase (Nichirei Biosciences Inc., Tokyo, Japan). Peroxidase activity
was visualized using 3,3′-diaminobenzidine tetrahydrochloride (Nacalai Tesque, Inc.,
Kyoto, Japan). Counterstaining was performed with hematoxylin and eosin (H & E).
Alcian Blue (pH2.5)/periodic acid-Schiff base (AB-PAS) staining was used for detection of
sugar chains attached to glycoproteins. Histochemical and biochemical analyses were
performed in at least three separate experiments. A group of five WT mice and a group of
five Nox1-/Y mice were used for each experiment. At least
three colon sections were analyzed, with at least three images examined for each. Twenty
crypts/colon were counted in analyses of histochemical damage, goblet cell damage, and
BrdU/Ki-67 staining. Colon crypt damage was evaluated by the presence of leukocyte
recruitment/infiltration, thickening of the colon wall, and loss or immaturity of goblet
cells, as described previously [21 (link)].
and retrieved as described previously [4 (link)]. The colon
sections were immunostained with various antibodies by using second antibodies conjugated
with horseradish peroxidase (Nichirei Biosciences Inc., Tokyo, Japan). Peroxidase activity
was visualized using 3,3′-diaminobenzidine tetrahydrochloride (Nacalai Tesque, Inc.,
Kyoto, Japan). Counterstaining was performed with hematoxylin and eosin (H & E).
Alcian Blue (pH2.5)/periodic acid-Schiff base (AB-PAS) staining was used for detection of
sugar chains attached to glycoproteins. Histochemical and biochemical analyses were
performed in at least three separate experiments. A group of five WT mice and a group of
five Nox1-/Y mice were used for each experiment. At least
three colon sections were analyzed, with at least three images examined for each. Twenty
crypts/colon were counted in analyses of histochemical damage, goblet cell damage, and
BrdU/Ki-67 staining. Colon crypt damage was evaluated by the presence of leukocyte
recruitment/infiltration, thickening of the colon wall, and loss or immaturity of goblet
cells, as described previously [21 (link)].
3
Immunohistochemical Detection of hTERT
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Serial sections were deparaffinized and immersed in methanol containing 0.3% (v/v) hydrogen peroxide for 15 min at room temperature to block endogenous peroxidase activity. After washing with running water and phosphate-buffered saline (PBS, pH 7.4), the sections were immersed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for 15 min at low power for antigen retrieval. Anti-human TERT (hTERT) rabbit polyclonal antibody (1:100 dilution; Millipore, Temecula, CA, USA) was applied to each section overnight at 4°C. The sections were then incubated with peroxidase-labeled dextran polymer (Simple Stain MAX-PO; Nichirei Bio, Tokyo, Japan) for 60 min at room temperature, and the reaction products were visualized by immersing the sections in freshly prepared 2 mM DAB solution (0.05% 3,3′-diaminobenzidine tetrahydrochloride; Nacalai Tesque, Kyoto, Japan) in 0.05 M Tris-HCl (pH 7.6) and 0.01% H 2 O 2 . Nuclei were lightly stained using Mayer's hematoxylin (Muto Pure Chemicals, Tokyo, Japan). A case was defined as positive when at least one cell showed a positive reaction.
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