Amomum xanthioides was obtained from an herbal pharmaceutical company (Jeong-Seong Drugstore, Daejeon, Rep. of Korea). After obtaining EFAX (at a final yield of 0.19% (w/w)), its chemical compositionwas analyzed using UHPLC/LC-MS analysis (See Figs S1 and S2 online). The reagents for the present study were as follows: Histofine (Nichirei Biosciences, Tokyo, Japan); hydrochloric acid and phosphoric acid (Kanto Chemical Co., Inc., Tokyo, Japan); n-butanol (J.T. Baker, Phillipsburg, NJ); diaminobenzidine (DAB) (Abcam, Cambridge, UK); Mayer’s hematoxylin, methanol and isopropanol (Wako Pure Chemical Industries, Osaka, Japan); TRI reagent (Invitrogen, Carlsbad, CA); goat anti-human CTGF antibody, CTFG standard solution, rabbit anti-human CTGF antibody, and anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA); TGF-β1 (R&D Systems, Minneapolis, MN); SB 525334 (TOCRIS Bioscience, Bristol, UK); and catechin, quercitrin, and procyanidin B2 (Kyeong-Buk, South Korea). All other materials were purchased from Sigma-Aldrich (St. Louis, MO).
Diaminobenzidine dab
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Diaminobenzidine (DAB) is a chromogenic substrate used in histochemistry and immunohistochemistry procedures. It is commonly used as a peroxidase substrate to visualize the presence of target antigens in biological samples.
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Lab products found in correlation
Avidin biotin peroxidase complex, by Vector Laboratories (5 mentions)
Primary mouse anti mouse mmp 9 antibody, by Abcam (2 mentions)
H e solution, by Merck Group (2 mentions)
Primary mouse antirabbit inos antibody, by Abcam (2 mentions)
Histofine, by Nichirei Biosciences (1 mentions)
Sb525334, by Bio-Techne (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
N butanol, by Avantor (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Nf b p65, by Abcam (1 mentions)
Rabbit anti irak1, by Abcam (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Avantor (1 mentions)
Anti rabbit biotinylated antibody, by Agilent Technologies (1 mentions)
Ab18976, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Horseradish peroxidase hrp secondary antibody, by Cell Signaling Technology (1 mentions)
13 protocols using diaminobenzidine dab
1
Phytochemical Analysis of Amomum xanthioides
2
Histological Analysis of Lung Inflammation
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The lung tissue was fixed in 4% (v/v) paraformaldehyde, embedded in paraffin, sectioned at 4-µm thickness, and stained with hematoxylin and eosin (H&E_solution; Sigma-Aldrich) to estimate inflammation.
Immunohistochemical slides were deparaffinized, dehydrated, washed in PBS containing 0.05% tween 20 (PBS-T), and incubated for 20 min at room temperature with goat serum to block nonspecific staining. The slides were incubated for 2 h at room temperature with primary mouse anti-mouse MMP-9 antibody (diluted 1:100, Abcam). After incubation, they were washed three times, incubated for 1 h at room temperature with a biotinylated secondary antibody, and then incubated with an avidinbiotin-peroxidase complex (Vector Laboratories, Burlin-game, CA, USA) for 1 h at room temperature. Then, the slides were washed with PBS-T and incubated with diaminobenzidine (DAB, Abcam) for an additional 5min.
Immunohistochemical slides were deparaffinized, dehydrated, washed in PBS containing 0.05% tween 20 (PBS-T), and incubated for 20 min at room temperature with goat serum to block nonspecific staining. The slides were incubated for 2 h at room temperature with primary mouse anti-mouse MMP-9 antibody (diluted 1:100, Abcam). After incubation, they were washed three times, incubated for 1 h at room temperature with a biotinylated secondary antibody, and then incubated with an avidinbiotin-peroxidase complex (Vector Laboratories, Burlin-game, CA, USA) for 1 h at room temperature. Then, the slides were washed with PBS-T and incubated with diaminobenzidine (DAB, Abcam) for an additional 5min.
3
Immunohistochemical Analysis of COX-2 Expression
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Briefly, formalin-fixed paraffin-embedded tissue sections were deparaffinized using xylene, rehydrated, and washed in phosphate-buffered saline (pH 7.0), and the remaining protocols were followed as per the method described earlier [26 (link),27 (link)]. The COX-2 protein of Abcam, Cambridge, U.K. was used as primary antibodies and incubated for 1 h at 4 °C, followed by incubation with the secondary antibody for 60 min, then streptavidin–biotin enzyme complex for 1 h. Diaminobenzidine (DAB) (Abcam, Cambridge, UK, ab 64259) chromogen was then applied accordingly, and hematoxylin was used as a counterstain. Finally, the results were analyzed under a light microscope.
4
Quercetin's Anti-Inflammatory Effects
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Quercetin was purchased from BOC Sciences (Shirley, NY, United States). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, United States). Primary antibodies (NF-B-p65, Nrf2, HO-1, cleaved caspase-3), secondary antibody biotinylated goat anti-mouse, and diaminobenzidine DAB were purchased from Abcam Co. (Waltham, MA, United States).
5
Immunohistochemical Analysis of IRAK1 Expression
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Tissue specimens were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Afterwards, sections were deparaffinized and rehydrated. Sections were treated with hydrogen peroxide for 10 min to block endogenous peroxide activity. After antigen retrieval using a microwave, the sections were treated with 1% bovine serum albumin to block non-specific binding. Sections were then incubated with rabbit anti-IRAK1 (Abcam, Cambridge, MA, USA) in a humidified chamber overnight at 4°C. After washing thrice with phosphate-buffered saline (PBS) for 5 min each wash, tissue sections were treated with biotinylated anti-rabbit secondary antibody (Abcam), followed by further incubation with streptavidin-horseradish peroxidase complex. After rinsing, sections were treated with diaminobenzidine (DAB; Abcam) as chromogen, and counterstained with hematoxylin. Samples incubated with PBS instead of the primary antibody served as negative controls.
6
Immunohistochemical Analysis of GFAP in Zebrafish
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Adult zebrafish from both eef1a2 mutant lines and age-matched wild-type controls were fixed in 10% neutral buffered formalin (Sigma–Aldrich). Spinal cord sections were cut at a thickness of 3 µm. Paraffin-embedded spinal cord sections were dewaxed with xylene and rehydrated through decreasing series of ethanol. Antigen retrieval was carried out using Proteinase K for 10 min at room temperature and slides were then treated with 3% hydrogen peroxide to block endogenous peroxidase. Sections were blocked with goat serum (1:5 in PBS) for 10 min. They were incubated overnight with anti-glial fibrillary acidic protein (GFAP) rabbit antibody (Dako) diluted at 1:500 in PBS, washed twice with PBS for 5 min and then incubated with anti-rabbit biotinylated antibody (Dako) at a concentration of 1:500 in PBS for 30 min at room temperature. Sections were then treated with Strept ABC reagent (Vector Laboratories) for 30 min and with Diaminobenzidine (DAB; Abcam) for 10 min. Sections were counterstained in Haematoxylin solution (Shandon), dehydrated and finally mounted in DPX (VWR).
7
Immunohistochemical Analysis of Lung Inflammation
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Fixed lung tissue samples were embedded in paraffin and sectioned into 4-µm-thick slices. The sections were stained with hematoxylin and eosin (H&E solution, Sigma-Aldrich) to evaluate inflammation. For the immunohistochemical assay, the paraffin-embedded lung tissues were deparaffinized, dehydrated, and washed in PBS containing 0.05% Tween 20 (PBS-T). The slides were incubated with goat serum for 20 min at room temperature to block nonspecific staining. They were then incubated for 2 h at room temperature with primary mouse antirabbit iNOS antibody (diluted 1:100, Abcam). Next, they were washed three times using PBS-T, incubated for 1 h at room temperature with a biotinylated secondary antibody, and again incubated with an avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. The slides were washed, incubated with diaminobenzidine (DAB, Abcam) for an additional 5 min, and examined using a microscope.
8
Histological Analysis of Lung Inflammation
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The lung tissue was fixed in 4% (v/v) paraformaldehyde, embedded in paraffin, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (H&E solution, Sigma–Aldrich) to estimate inflammation.
Immunohistochemical slides were deparaffinized, dehydrated, washed in PBS containing 0.05% tween 20 (PBS-T), and incubated for 20 min at room temperature with goat serum to block non-specific staining. The slides were incubated for 2 h at room temperature with primary mouse anti-mouse MMP-9 antibody (diluted 1:100, Abcam). After incubation, they were washed three times, incubated for 1 h at room temperature with biotinylated secondary antibody, and then incubated with an avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA, United States) for 1 h at room temperature. Then, the slides were washed with PBS-T and incubated with diaminobenzidine (DAB, Abcam) for an additional 5 min.
Immunohistochemical slides were deparaffinized, dehydrated, washed in PBS containing 0.05% tween 20 (PBS-T), and incubated for 20 min at room temperature with goat serum to block non-specific staining. The slides were incubated for 2 h at room temperature with primary mouse anti-mouse MMP-9 antibody (diluted 1:100, Abcam). After incubation, they were washed three times, incubated for 1 h at room temperature with biotinylated secondary antibody, and then incubated with an avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA, United States) for 1 h at room temperature. Then, the slides were washed with PBS-T and incubated with diaminobenzidine (DAB, Abcam) for an additional 5 min.
9
Lung Histopathology and Immunohistochemistry
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The mouse right lungs were fixed in a 10% neutral buffered formalin and embedded to make in paraffin blocks, which were then sectioned into 4 μm thick slices. The sections were then stained with hematoxylin and eosin (H&E) to evaluate inflammatory responses in lung tissue. In addition, the lung tissues were stained with priodic acid-schiiff (PAS) to detect mucus production. Quantitative analysis of airway inflammation and mucus production was determined using an image analyzer (IMT i-solution software, Vancouver, BC, Cananda).
For immunohistochemical analysis, sections were deparaffinized, dehydrated, and washed in PBS containing 0.05% tween 20 (PBS-T). The slides were incubated to block nonspecific staining for 20 min at room temperature with goat serum. The slides were incubated with primary mouse anti-mouse iNOS antibody (diluted 1:100, Abcam, Cambridge, UK) for 2 h at room temperature. After incubation, they were incubated for 1 h at room temperature with biotinylated secondary antibody, and then incubated with an avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Then, the slides were washed with PBS-T and incubated with diaminobenzidine (DAB, Abcam) for an additional 5 min.
For immunohistochemical analysis, sections were deparaffinized, dehydrated, and washed in PBS containing 0.05% tween 20 (PBS-T). The slides were incubated to block nonspecific staining for 20 min at room temperature with goat serum. The slides were incubated with primary mouse anti-mouse iNOS antibody (diluted 1:100, Abcam, Cambridge, UK) for 2 h at room temperature. After incubation, they were incubated for 1 h at room temperature with biotinylated secondary antibody, and then incubated with an avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Then, the slides were washed with PBS-T and incubated with diaminobenzidine (DAB, Abcam) for an additional 5 min.
10
Immunohistochemical Analysis of Oct-4 in Tissue
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The tissue sections were initially fixed in 10% formalin solution at 42°C for 2 days and paraffin embedded. The tissue sections were subsequently subjected to microtome sectioning (5 µm). The sections were placed on glass slides, de-paraffinized and rehydrated. The endogenous peroxidase activity was blocked by immersing the sections in freshly prepared 10% H2O2 and 10% methanol in 1X phosphate-buffered saline (PBS) for 20 min. The sections underwent trypsin treatment (0.1% trypsin in 0.1% CaCl2) for 10 min to cleave the protein crosslinks to assess the antigen and epitope. Nonspecific antigens were blocked using 4% bovine serum albumin (BSA; Sigma-Aldrich; Merck KgaA, Darmstadt, Germany) for 2 h at room temperature. The membranes were incubated with an anti-Oct-4 primary antibody (dilution, 1:100; cat no. ab18976; Abcam, Cambridge, UK) overnight at 4°C. Following incubation, the sections were thoroughly washed with 1X PBS and incubated with a goat anti-rabbit secondary antibody (dilution, 1:3,000; cat no. ab6721; Abcam) for 1 h at room temperature. Following washing to prevent non-specific binding, the sections were stained with diaminobenzidine (DAB; cat no. ab64238; Abcam).
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