Tissue non-heme iron was stained according to previously published protocols49 (link). Sections of fixed, frozen human tissue was allowed to warm to room temperature and dried for 15 minutes in a laminar flow hood. Endogenous peroxidase activity was quenched by immersion in a solution of 0.3% H2O2 (v/v) in methanol for 20 minutes and washed three times in deionized water (dH2O). Sections were then placed in a solution of fresh 1% (w/v) potassium ferrocyanide (Sigma-Aldrich, UK), pH 1 with HCL for 40 minutes, followed by three washes in dH2O. Sections were then placed in 0.01M NaN3, 0.3% H2O2 in methanol for 60 minutes, followed by three washes in PBS. Iron staining was intensified using 3’-diaminobenzidine (DAB) (10% v/v) solution from Pierce DAB substrate kit (Thermo Fisher) in PBS with 0.005% H2O2 (v/v) for five hours. DAB reaction was halted with three washes in PBS, 1 wash in 100% methanol and a further three washes in Bond Wash solution (Leica Biosystems).
Pierce dab substrate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce™ DAB Substrate Kit is a reagent used in immunohistochemistry and immunocytochemistry applications to visualize target proteins. The kit contains the necessary components to facilitate a chromogenic reaction that produces a brown precipitate, allowing for the detection and localization of the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (3 mentions)
Bond wash solution, by Leica (2 mentions)
Potassium ferrocyanide, by Merck Group (2 mentions)
Chamber slide, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Alexa flour 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Animal free blocking solution, by Cell Signaling Technology (1 mentions)
Signalstain boost detection reagent, by Cell Signaling Technology (1 mentions)
Citrate buffer ph 6, by Merck Group (1 mentions)
Ab97051, by Abcam (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
32 protocols using pierce dab substrate kit
1
Tissue Non-Heme Iron Staining
2
Quantifying HCV Infection in Huh7.5 Cells
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Huh7.5 cells (obtained from Apath, LLC; New York, USA) were maintained in DMEM (Gibco) with 10% fetal bovine serum (Sigma) and penicillin (100 U/mL) / streptomycin (100 µg/mL) (Sigma) and were incubated at 37 °C and 5% CO2. Adenovirus Expression Medium (AEM) (Gibco), supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL), was used for HCV production under serum-free conditions38 (link).
The percentage of HCV infected cells was evaluated by immunostainings38 (link),43 (link). In brief, cells were seeded in a chamber slide (Thermo Fisher Scientific) for a confluent cell layer, fixed with acetone (Merck) the next day, and stained with primary antibody 9E10 diluted 1:3,00044 (link), followed by secondary antibody Alexa Flour 488 goat anti-mouse IgG diluted 1:500 (Invitrogen), and Hoechst 33342 (Molecular Probes) diluted 1:1,000.
HCV-infectivity titres were determined with three technical replicates as FFU/mL in a cell-based assay in 96-well plates as described45 (link),46 (link). The immunostaining of 96-well plates was carried out with primary antibody 9E10 diluted 1:5,000, secondary antibody ECL Anti-mouse IgG Horseradish Peroxidase linked from sheep (Amersham Biosciences) diluted 1:500, and visualized with Pierce DAB Substrate Kit (Thermo Scientific). 96-well plates were imaged and automatically counted for FFU quantification.
The percentage of HCV infected cells was evaluated by immunostainings38 (link),43 (link). In brief, cells were seeded in a chamber slide (Thermo Fisher Scientific) for a confluent cell layer, fixed with acetone (Merck) the next day, and stained with primary antibody 9E10 diluted 1:3,00044 (link), followed by secondary antibody Alexa Flour 488 goat anti-mouse IgG diluted 1:500 (Invitrogen), and Hoechst 33342 (Molecular Probes) diluted 1:1,000.
HCV-infectivity titres were determined with three technical replicates as FFU/mL in a cell-based assay in 96-well plates as described45 (link),46 (link). The immunostaining of 96-well plates was carried out with primary antibody 9E10 diluted 1:5,000, secondary antibody ECL Anti-mouse IgG Horseradish Peroxidase linked from sheep (Amersham Biosciences) diluted 1:500, and visualized with Pierce DAB Substrate Kit (Thermo Scientific). 96-well plates were imaged and automatically counted for FFU quantification.
3
Immunohistochemistry and Immunofluorescence Staining Protocol
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For IHC staining, the sections were dewaxed 3 times with xylene for 10 minutes each, rehydrated, and then microwaved for 20 minutes with citrate buffer pH 6.0 (Sigma-Aldrich) to retrieve the epitope, as described previously (2 (link)). The sections were incubated with animal-free blocking solution (Cell Signaling Technology) for 1 hour, washed in PBS, and then incubated with primary antibodies overnight at 4°C. After 3 washes with PBS, the sections were incubated with the Signal Stain Boost Detection Reagent (Cell Signaling Technology) for 1 hour at room temperature. The tissue sections were then washed 3 times with PBS, detected using a Pierce DAB Substrate Kit (Thermo Fisher Scientific), stained with hematoxylin, and imaged by confocal microscopy. For immunofluorescent staining, the tissue sections were incubated with primary antibodies overnight at 4°C, washed, and incubated with Alexa Fluor–labeled secondary antibodies for 1 hour at room temperature. After PBS washing, both cells and tissue sections were incubated with diluted DAPI solution for 5 minutes at room temperature, mounted with an antifade mounting media, and imaged by confocal microscopy. All antibodies are listed in Supplemental Table 4 .
4
Western Blot Analysis of Protein Expression
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The procedure for immunoblotting has been previously described (16 (link)). Cells were treated with cell lysis buffer and then protein samples were collected. Briefly, 40 µg protein/lane was loaded into the lanes of 12% denatured polyacrylamide gel. The proteins were separated by electrophoresis and transferred to nitrocellulose membranes (0.45 µm; Schleicher & Schuell; GE Healthcare Life Sciences). The membranes were blocked with 3% bovine serum albumin in 0.01 M PBS (pH 7.4) and 0.05% Tween-20 (PBST) at room temperature for 1 h. Subsequently, the membrane was incubated with the aforementioned primary antibodies against the target proteins overnight at 4°C. Following two quick washes in PBST, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase [Goat Anti-Rabbit IgG H&L (HRP); dilution, 1:4,000 in PBST; ab97051; Abcam, Cambridge, UK) for 2 h at room temperature. Following washes as aforementioned, an enhanced chemiluminescence reagent (Pierce™ DAB Substrate kit; 34002; Thermo Fisher Scientific, Inc.) was used to produce fluorescence for detection. The density of immunoblotting was quantified using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative expression of the target protein was quantified as a ratio against GAPDH.
5
Tissue Non-Heme Iron Staining
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Tissue non-heme iron was stained according to previously published protocols49 (link). Sections of fixed, frozen human tissue was allowed to warm to room temperature and dried for 15 minutes in a laminar flow hood. Endogenous peroxidase activity was quenched by immersion in a solution of 0.3% H2O2 (v/v) in methanol for 20 minutes and washed three times in deionized water (dH2O). Sections were then placed in a solution of fresh 1% (w/v) potassium ferrocyanide (Sigma-Aldrich, UK), pH 1 with HCL for 40 minutes, followed by three washes in dH2O. Sections were then placed in 0.01M NaN3, 0.3% H2O2 in methanol for 60 minutes, followed by three washes in PBS. Iron staining was intensified using 3’-diaminobenzidine (DAB) (10% v/v) solution from Pierce DAB substrate kit (Thermo Fisher) in PBS with 0.005% H2O2 (v/v) for five hours. DAB reaction was halted with three washes in PBS, 1 wash in 100% methanol and a further three washes in Bond Wash solution (Leica Biosystems).
6
Vimentin Immunohistochemical Staining
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Cells were harvested from plates with trypsin (Corning), spun down, resuspended in PBS, smeared, air-dried and fixed in acetone. For antigen retrieval, slides were placed in sodium citrate buffer (pH 6.0) in a pressure cooker for 2 min in a microwave oven at full power. A 3% peroxidase blocking solution (30% H2O2 in methanol) was used to reduce endogenous peroxidase activity. After peroxidase quenching, slides were blocked for 30 min at room temperature using a homemade blocking reagent (4% BSA and 0.02% Tween). Primary immunostaining (Anti-Vimentin antibody (1:100), Abcam ab8069, Cambridge, MA) was performed for 7 min at 37 °C. Secondary biotinylated antibody and detection steps were followed according to manufacturer’s instructions (VECTASTAIN® Elite® ABC-HRP Kit Peroxidase, Universal, PK-6200, Vector Laboratories, Inc., Burlingame, CA). The chromogenic reaction was accomplished using the Pierce™ DAB Substrate Kit (Cat#34002 Thermo Fisher Scientific, Rockford, IL). Hematoxylin was used as a counterstain.
7
Free-Floating Immunohistochemistry Protocol
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Free-floating immunohistochemistry was done after pretreatment (1% peroxide in ice cold methanol for 20 min, 0.3% glycine in phosphate buffered saline (PBS) for 10 min and 0.03% SDS in PBS for 10 min) and blocking (3% normal donkey serum in PBS/0.25% Triton X-100 for 1–3 h). All primary and secondary antibodies were diluted in blocking solutions. Primary antibodies were incubated at 4 °C at least over night or for 48 h and secondary antibodies were incubated at room temperature for 2 h. For rabbit anti-pSTAT3 (1:1000, Cell Signaling) and rabbit anti-cFos (1:5000, Millipore/Calbiochem) staining was developed with ABC/3,3′ diaminobenzidine tetrahydrochloride (Pierce DAB Substrate Kit, ThermoScientific) as described earlier [69] (link). Double labeling was done with either DsRed (1:1000, rabbit anti-DsRed, Santa Cruz), WGA (1:1000, goat anti-WGA, Vector Laboratories), GFP (1:000, chicken anti-GFP; AbCAM), or orexin/hypocretin-A (1:1000, goat anti-orexin/hypocretin-A, Santa Cruz) was visualized with fluorophor-labeled secondary antibodies (1:200 Alexa 568 or 488, Invitorgen or 1:400 Dylite, Jackson ImmunoResearch). Sections were mounted onto gelatin-subbed slides and cover slipped with ProLong Anti-fade mounting medium (Invitrogen) for further microscopy analysis.
8
Immunohistochemical Analysis of Proteins
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For the immunohistochemistry assay, the prepared sections were submerged in citrate buffer (pH 6.0) and heated at boiling temperature in a pressure cooker for 10 min for antigen retrieval. The expression level of Htt, GTPCH and DHFR proteins were examined in each group after incubation with primary antibody overnight and HRP-conjugated secondary antibody incubation at 37 °C for 45 min. The reaction was visualized with a Pierce™ DAB Substrate Kit (Thermo Fisher Scientific) followed by hematoxylin staining (Abcam) of the nuclei. The number of positively stained cells was counted in five randomly selected microscopic fields at 400 ×. The average of stained cells was calculated.
9
Immunohistochemical Detection of Sphingosine
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Samples were cut at 6 μm, deparaffinized, rehydrated, and boiled with citrate buffer (BioLegend; #420902) for 10 min. After cooling, the samples were treated with 0.3% hydrogen peroxide for 10 min. Next, they were washed twice with PBS and blocked for 10 min at room temperature with PBS, 0.05% Tween 20 (Sigma, St. Louis, MO, USA), and 5% fetal calf serum (FCS). Samples were stained with anti-sphingosine (1:100 dilution, Cosmobio, Tokyo, Japan; #ALF-274042010) in PBS, 0.05% Tween 20, and 1% FCS at 4 °C overnight. The samples were washed three times with PBS plus 0.05% Tween 20. We secondary-labeled the tissues for 45 min at room temperature with ZytoChem Plus (HRP) One-Step Polymer anti-mouse/rabbit (Zytomed Systems, Berlin, Germany; #ZUC053-100) in PBS, 0.05% Tween 20, and 1% FCS. The tissues were washed, then the DAB substrate (Pierce DAB-Substrate Kit, Thermo Fischer Scientific, Waltham, MA, USA; #34002) was added to the samples for 4 min at room temperature. The samples were counterstained with hematoxylin for 2 min. Finally, the tissues were dehydrated with ethanol to xylene and mounted with Eukitt.
10
Eimeria Sporozoite Protein Profiling
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Twenty micrograms of protein samples from co-cultured jejunal epithelial cells were subjected to SDS-PAGE using a routine method described elsewhere [13 (link)].
Western blot was employed to analyze the soluble proteins of E. maxima sporozoite that bound to chicken jejunal epithelial cells. Briefly, the protein samples were separated using a 12% SDS-PAGE gel as described above and transferred to a nitrocellulose membrane (Millipore) using Semi-Dry Transfer Cell (Bio-Rad, Hercules, USA) according to the manufacturer’s instructions. Rat antiserum against the soluble proteins of E. maxima sporozoite (dilution 1:200) was used as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (dilution 1:2000, Sigma) was used as the secondary antibody. The bound antibodies were detected using Pierce DAB Substrate Kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions.
Western blot was employed to analyze the soluble proteins of E. maxima sporozoite that bound to chicken jejunal epithelial cells. Briefly, the protein samples were separated using a 12% SDS-PAGE gel as described above and transferred to a nitrocellulose membrane (Millipore) using Semi-Dry Transfer Cell (Bio-Rad, Hercules, USA) according to the manufacturer’s instructions. Rat antiserum against the soluble proteins of E. maxima sporozoite (dilution 1:200) was used as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (dilution 1:2000, Sigma) was used as the secondary antibody. The bound antibodies were detected using Pierce DAB Substrate Kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions.
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