The aforementioned 3-µm-thick kidney tissue sections were stained immunohistochemically with antibodies against TGF-β1 (1:1,000; cat. no. ab92486, Abcam), Smad3 (1:500; cat. no. ab40845; Abcam) and α-SMA (1:500; cat. no. bs-10196R; BIOSS). After deparaffin (xylene and gradient ethanol) and rehydration, slices were boiled for 15 min using microwave irradiation for antigen retrieval in citrate buffer (0.01 mol/l; pH 6.0). Slices were then washed three times with PBS and blocked with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. The slides were incubated with the aforementioned primary antibodies for 2 h at room temperature. Goat anti-rabbit IgG peroxidase conjugate (1:500; cat. no. bs-0295G, BIOSS) was used as the secondary antibody. Cells were incubated with secondary antibodies for 1 h and room temperature. 3,39-diaminobenzidine, nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (all, Sigma-Aldrich; Merck KGaA) were used as enzyme substrates. Finally, after dehydration with gradient ethanol and permeabilization with xylene, the slides were sealed and photographed. Visual analysis was performed using an Olympus inverted light microscope (magnification, ×400; cat. no. CX71; Olympus Corporation). The mean optical density (MOD) was calculated using Image Pro Plus 6.0 software (Media Cybernetics, Inc.).
5 bromo 4 chloro 3 indolyl phosphate
Manufactured by Merck Group
Sourced in United States
5-bromo-4-chloro-3-indolyl phosphate is a chromogenic substrate used in biochemical and molecular biology applications. It is a colorless compound that, when hydrolyzed by alkaline phosphatase, produces a blue-colored precipitate.
Automatically generated - may contain errors
Lab products found in correlation
Nitroblue tetrazolium, by Merck Group (28 mentions)
Nitroblue tetrazolium chloride, by Merck Group (4 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Protein assay reagents with bovine albumin, by Bio-Rad (2 mentions)
Glomax spectrophotometer, by Promega (2 mentions)
Inverted light microscope, by Nikon (2 mentions)
Streptavidin alkaline phosphatase, by BD (2 mentions)
Goat anti rabbit antibody conjugated with alkaline phosphates, by Merck Group (2 mentions)
N n dimethylformamide (dmf), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Ab92486, by Abcam (1 mentions)
Ab40845, by Abcam (1 mentions)
Bs 10196r, by Bioss Antibodies (1 mentions)
Inverted light microscope, by Olympus (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Streptavidin alkaline phosphatase, by Merck Group (1 mentions)
Anti igg or igm biotin, by Southern Biotech (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Calf thymus histone, by Roche (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
57 protocols using 5 bromo 4 chloro 3 indolyl phosphate
1
Immunohistochemical Analysis of Kidney Fibrosis
2
Hfq Protein Detection in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The OMV contents purified from WT E. coli cells were analyzed via Western blotting (WB). The WB membrane was revealed as described previously [62 (link)]; briefly, the membrane was successively incubated with a goat anti-Hfq polyclonal antibody for one hour at room temperature (dilution 1/1000, Origene, Herford, Germany). Then, after extensive washing, the goat antibody was revealed with an anti-goat secondary antibody coupled to alkaline phosphatase for one hour at room temperature (dilution 1/1000, Sigma), and was revealed with an NBT/BCIP solution (nitro blue tetrazolium chloride and 5-Bromo-4-Chloro-3-Indolyl-Phosphate, Sigma). We previously showed that the commercial antibodies for goat are specific using a Δhfq strain [62 (link)].
3
Enzyme-Linked Immunospot Assay for Antibody-Secreting Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ninety-six-well plates were coated with 20 μg ml−1 NP22CGG or OVA, 512 HA units per mL PR8 virus, or 10 μg ml−1 LCMV virus and blocked with complete RPMI (10% fetal bovine serum, 1% penicillin/streptomycin, 1% HEPES and 50 μM 2-mercaptoethanol). Bone marrow and splenic cells isolated from immunized mice were serially diluted in complete RPMI and incubated 16 h at 37 °C. The plates were then treated with either anti-IgG- or -IgM-biotin (Southern Biotechnology) followed by incubation with streptavidin-alkaline phosphatase (Sigma). Plates were then developed with 5-bromo-4-chloro-3-indolylphosphate (Sigma) until spots developed and spots counted with CTL Immunospot software.
4
Alkaline Phosphatase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The differentiated cells were fixed with 4% formaldehyde (Sigma-Aldrich, St Louis, MO, USA) for 30 min. After washing with 2 mM MgCl2 solution, cells were incubated with alkaline phosphatase (AP) buffer (100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 10 mM MgCl2, and 0.05% Tween-20) for 15 min. They were then incubated in AP buffer containing 0.4 mg/mL nitro blue tetrazolium (Sigma-Aldrich, St Louis, MO, USA) and 0.2 g/mL of 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich, St Louis, MO, USA). The reaction was terminated with 5 mM EDTA solution (pH 8.0). The degree of staining was measured at 405 nm using a SpectraMax M2 microplate spectrophotometer.
5
Quantification of Autoantibodies in Murine Lupus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum samples were taken from mice at the beginning (eight weeks) and end (15 weeks) of the experiments and were stored at -35°C until anti-dsDNA (15 (link)), and anti-Histone (22 (link)) antibodies were analyzed by ELISA as follows. A MaxiSorp plate (Nunc, Rochester, NY, USA) from 96-well was coated with 2.5 μg/ml calf thymus dsDNA (Sigma Aldrich, St Louis, MO, USA) or 10 μg/ml calf thymus histone (Roche Diagnostic, Mannheim, Germany) in 100 μl of bicarbonate buffer overnight at 4°C. Bovine serum albumin (BSA, Invitrogen, Carlsbad CA, USA) at 2% was used to block the plate. The plate was incubated for one hour at 37°C with serum (1:50) or the anti-dsDNA antibody standard (clone 16-13, Chemicon International, Billerica MA, USA) or was incubated for two hours at room temperature with serum (1:150) for anti-histone antibody. The plate was washed and incubated with rabbit anti-mouse IgM, IgG, IgG1, or IgG2a conjugated to alkaline phosphatase (AP, Zymed Laboratories, San Francisco CA, USA) or anti IgG2b or IgG3 conjugated to peroxidase (HRP), after which substrate was added. (5-bromo-4-chloro-3- indolyl phosphate; Sigma−Aldrich, St Louis MO, USA) for AP or HRP substrate (3.3’,5,5’ tetramethylbenzidine TMB Sigma−Aldrich), respectively. The O.D. was read at 405 or 450 nm using a Dynatech MR5000 ELISA reader.
6
AAV-Mediated Transduction in Mouse Lungs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C57BL/6 mice (Jackson Labs) were administered 1 × 1011 vg of AAV6-AP or AAV6.2FF-AP by a modified intranasal technique as described previously.23 (link) Three weeks following AAV delivery, the lungs and nose were harvested and fixed in 2% paraformaldehyde for 2 or 16 hr, respectively. Tissues were washed three times in PBS and heat-inactivated for 1 hr at 65°C prior to overnight incubation in AP staining buffer (100 mM Tris [pH 8.5], 100 mM NaCl, 50 mM MgCl2) with 100× X-PHOS (10 mg/mL 5-bromo-4-chloro-3-indolyl phosphate; Sigma) and 100× nitro blue tetrazolium chloride (50 mg/mL; Invitrogen). Gross images were acquired prior to paraffin embedding and sectioning for histological staining. Transduction was quantified by analyzing the copy number of AAV vg per 100 ng of genomic DNA extracted from paraffin-embedded tissue by qPCR as previously described.23 (link) Quantification of AP present in homogenized lung tissue was performed as previously reported.21 (link)
7
ELISPOT Assay for Anti-OVA Antibodies
8
Alkaline Phosphatase Staining for Osteoblast Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ALP activity was analyzed after 7 days of culture. Cell layers were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min at 4 °C, rinsed with PBS, and stained with 5-bromo-4-chloro-3-indolyl phosphate (catalog no. B5655-5TAB, Sigma-Aldrich, St. Louis, MO, USA) for 2 h in darkness at room temperature. The chromogenic reactions were stopped by washing the samples twice with dH2O. Following drying, the samples were observed under a light microscope (CKX31, Olympus, Tokyo, Japan) at ×40 magnification. Alkaline phosphatase activity due to osteoblast differentiation was stained a dark purple color. Cells exhibiting ALP activity were identified by culture wells stained a purple-blue color. The stained percent (%) within 6 mm2 of the observation area was analyzed once for each sample (n = 4) with ImageJ.
9
TUNEL Staining of Apoptotic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detects DNA breaks formed during apoptosis. Whole mount TUNEL staining of developmentally staged (neurula) control and morphant embryos was carried out as described in [77 (link)] with the following reagents: terminal deoxynucleotidyl transferase (TdT) (15 U/μl and 5x buffer, Thermofisher Ref. 10533065), digoxigenin-11-dUTP (25 nmol/25μl, Sigma Ref. 11093088910), Normal goat serum (Thermofisher Ref. 10000C), anti-dig alkaline phosphatase conjugate (Sigma Ref. 11093274910), nitro blue tetrazolium (Sigma Ref. 11383213001), and 5-bromo-4-chloro-3-indolyl phosphate (Sigma Ref. 10760994001). The control DMRT5 transcript was prepared as previously described [50 (link)].
10
Protein Extraction, Separation, and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted and separated using the Mini-Protein TGX gradient gel (4%–15%) as previously described (Le Roux et al., 2019 (link)). The Bio-Rad protein assay reagents with bovine albumin as the standard (Bio-Rad Laboratories Inc., Hercules, CA) was used for determination of protein concentration (Bradford, 1976 (link)), and quantified using a Glomax Spectrophotometer (Promega, Sunnyvale, CA) (Rylutt and Parish, 1982 (link)).
Western blot analyses were conducted using a Bio-Rad Trans-Blot® SD semi-dry transfer cell apparatus and polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences). The membranes were blocked with 3% bovine serum albumin (BSA) and probed with polyclonal large (RbcL) and small (RbcS) RuBisCO Subunits (RbcL and RbcS, 1:50000; Botha and Small, 1987 (link)) and human anti-SUMO1 monoclonal antibody (1:2500) (UBPBio, Aurora, USA) diluted in buffered saline containing 3% BSA. Detection employed alkaline phosphatase conjugated Donkey Anti-Mouse (Abcam) (1:2500) or goat anti-rabbit (1:7000) (Sigma-Aldrich, St. Louis, MO, USA) antibodies in conjunction with nitro blue tetrazolium and 5-Bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich, St. Louis, MO, USA).
Western blot analyses were conducted using a Bio-Rad Trans-Blot® SD semi-dry transfer cell apparatus and polyvinylidene difluoride membranes (Hybond-P, Amersham Biosciences). The membranes were blocked with 3% bovine serum albumin (BSA) and probed with polyclonal large (RbcL) and small (RbcS) RuBisCO Subunits (RbcL and RbcS, 1:50000; Botha and Small, 1987 (link)) and human anti-SUMO1 monoclonal antibody (1:2500) (UBPBio, Aurora, USA) diluted in buffered saline containing 3% BSA. Detection employed alkaline phosphatase conjugated Donkey Anti-Mouse (Abcam) (1:2500) or goat anti-rabbit (1:7000) (Sigma-Aldrich, St. Louis, MO, USA) antibodies in conjunction with nitro blue tetrazolium and 5-Bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich, St. Louis, MO, USA).
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!