Immunocytochemistry was used to detect the presence of PCNA and bax in the cells, as described previously [19 (link),20 (link),57 (link),58 (link),59 (link),60 (link),61 (link)], by means of primary monoclonal antibodies against PCNA and bax (dilution 1:500; from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), secondary swine antibodies against mouse IgG labeled with horseradish peroxidase (dilution 1:1000; Servac, Prague, Czech Republic) and visualized by DAB-substrate staining (Roche Diagnostics GmbH, Manheim, Germany). In some cases, the assay was validated by these primary antibodies and secondary polyclonal goat antibodies against mouse IgGs, labeled with fluorescein isothiocyanate (FITC, dilution 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; Figure 1 ). The negative controls were performed with cells processed without the primary or secondary antibody. The cells were inspected with the aid of a light and a fluorescence microscope (Leica GmbH, Wetzlar, Germany). The cells showing a signal larger than that of the levels of the background negative controls were considered positive. The percentage of cells containing a visible signal–marker of PCNA and bax was calculated relative to the total cell number.
Primary monoclonal antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Primary monoclonal antibodies are immunoglobulin molecules produced by a single clone of B cells. They recognize a specific epitope or antigen and are widely used in various laboratory applications, such as immunoassays, immunohistochemistry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein isothiocyanate fitc, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl system, by Cytiva (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Pvdf membrane, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Caspase 3, by Santa Cruz Biotechnology (1 mentions)
Trans blot, by Bio-Rad (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase labeled secondary goat anti mouse igg antibody, by Santa Cruz Biotechnology (1 mentions)
3h dexamethasone, by Cytiva (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Fluorescein isothiocyanate (fitc), by Santa Cruz Biotechnology (1 mentions)
Dab substrate, by Roche (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Infrared imaging system, by LI COR (1 mentions)
β actin, by Abcam (1 mentions)
9 protocols using primary monoclonal antibody
1
Immunocytochemical Detection of PCNA and Bax in Cells
2
Western Blot Analysis of Protein Lysates
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U266 cells of each group were collected and washed in lysis buffer. Total protein lysates from cells was extracted and western blot analyses were performed as described previously 18 (link). Blots were incubated respectively with different primary monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight and incubated subsequently with corresponding horseradish peroxidase conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) for 2 hours at room temperature. Enhanced chemiluminescence (ECL system; Amersham, UK) were used to visualize protein bands and Image J software (NIH, USA) were employed to quantify densitometry.
3
Quantifying Protein Levels in Mouse Brains
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Western blotting was carried out according to the method reported by Badshah et al. [44 (link)] with minor alterations. The mice brains were collected as quickly as possible and then homogenized in a T-PER solution. The levels of proteins in all 4 groups were measured using a BioRad protein assay. For each of the 4 groups, 30 μg of the protein content was run on a 15–20% SDS-PAGE. After completing the electrophoresis process, all the proteins were shifted to a PVDF membrane (Santa Cruz Biotech, USA) over transblot (Bio-Rad). Different primary monoclonal antibodies (Santa Cruz, CA, USA) such as caspase-3, Bcl-2-associated X protein (BAX), beta-secretase-1 (BACE-1), poly (ADP-ribose) polymerase-1 (PARP-1), amyloid beta (Aβ), synaptophysin (SYP), postsynaptic density protein 95 (PSD-95), and beta actin as well as HRP-conjugated secondary antibodies (Santa Cruz, CA, USA) were smeared. All results were visualised in a dark room on X-ray films.
4
Glucocorticoid Receptor Pathway Investigation
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Primary monoclonal antibodies against GRα, GRβ, phospho-p38MAPK, phospho-JNK and β-actin, as well as a horseradish peroxidase-labeled secondary goat anti-mouse IgG antibody, were purchased from Santa Cruz Biotechnology (USA). IL-1β was purchased from PeproTech (England) and RPMI1640 culture medium was purchased from GIBCO (USA). The specific inhibitors of the p38 MAPK pathway (SB203580), ERK pathway (PD98059), PI3K pathway (LY294002), PKC pathway (Ro31-8220) and JNK pathway (SP600125) were all purchased from Calbiochem (USA). [3H]-Dexamethasone was obtained from Amersham (USA). Dexamethasone was purchased from Sigma (USA). The primers and Taqman probes targeting GRα, GRβ, p38 MAPK and JNK were designed and synthesized by Applied Biosystems (USA). All other chemicals used in the study were of analytical grade and obtained from commercial sources.
5
Protein Expression Regulation by 5′ UTR and siRNA
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For protein expression of GFP and GAPDH under the influence of 5′ UTR and siRNA, the same transfection protocol was repeated (as described in 2.8) in 6-well plates with 0.5 µg of constructed vector and 40 nM/well of each siRNA. Un-transfected and siRNA transfected Huh-7 cell lysates were subjected to 12% SDS-PAGE at 24 and 48 h post-transfection by following a standard Western blotting protocol as previously described (Shahid et al., 2017 (link), Shahid et al., 2018 (link)). The protein expression incubated against primary monoclonal antibodies (Santa Cruz Biotechnology Inc, USA) specific to GFP and GAPDH, and secondary horseradish per-oxidase-conjugated anti-goat anti-mouse antibodies (Sigma Aldrich, USA) were evaluated by using Chemiluminescence’s detection kit (Sigma Aldrich, USA).
6
Quantifying Nuclear Translocation of NF-κB
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Cells were harvested, lysed in RIPA buffer (Beyotime Biotech, Jiangsu, China), and the nuclear and cytoplasmic proteins were extracted, followed by the determination of their concentrations using a BCA protein assay kit (Pierce, Rockford, IL). The protein sample was separated by 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After being blocked with 5% non-fat milk, the membrane was incubated with the primary monoclonal antibodies (all from Santa Cruz, CA) against MMP-2 (#sc-13594), MMP-9 (#sc-393859), NF-κB/p65 (#sc-8008), and GAPDH (#sc-47724) at 4°C overnight. Two hours after incubation with the second antibody, the membrane was rinsed with PBS and visualized with enhanced chemiluminescence on X-ray film. For expression of MMP-2 and MMP-9, results were expressed as the ratio of the optical density of the interested band divided by that for GAPDH band. Levels of NF-κB/p65 between nuclear and cytoplasmic fractions were compared to evaluate the nuclear translocation activity of NF-κB.
7
Immunocytochemistry for PCNA and Bax
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The presence of PCNA and bax in the cells was detected by immunocytochemistry, as described previously elsewhere (Roychoudhury et al., 2014; (link)Sirotkin et al., 2015) , by using primary monoclonal antibodies against these molecules (all from Santa Cruz Biotechnology, Inc., Santa Cruz, USA). They were placed in either, a dilution of 1:500 in PBS secondary swine antibodies against mouse IgG labeled with horseradish peroxidase (Servac, Prague, Czech Republic, dilution of 1:1000) and visualized by staining with DAB-substrate (Roche Diagnostics GmbH, Manheim, Germany), or by secondary polyclonal goat antibodies against mouse IgGs labeled with the fluorescent marker fluorescein isothiocyanate (FITC; Santa Cruz Biotechnology, dilution 1:1000). The presence of molecules in the cells was determined using a light and fluorescence microscope (Leica GmbH, Wetzlar, Germany). Cells processed without the primary or secondary antibody were used as the negative controls. The cells expressing a signal greater than the background negative control levels were considered positive. The proportion of cells containing visible molecules relative to the total cell number was calculated.
8
Western Blot Analysis of TCRζ and CD3ε
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Enriched T cells (5 × 106) of blood origin were used. Cells were pelleted and resuspended in 50 μL of lysis buffer supplemented with protease inhibitors, 5 mM NaF, 0.2 mM sodium vanadate, and 1 mM phenylmethylsulfonyl fluoride (PMSF) for two hours on ice. Then, the suspension was centrifuged at 14000 rpm for 5 minutes, and the supernatant collected. 40 μg of protein extract were boiled (100°C, 5 min) and resolved on a 12% polyacrylamide gel in reducing conditions (100 V, 90 min) and transferred (30 V, 60 min) onto a polyvinylidene fluoride (PDVF) membrane. Membrane was then blocked in nonfat milk, followed by incubation with a primary monoclonal antibody (Santa Cruz) TCRζ- or a CD3ε-specific (overnight incubation, 4°C). After several washing steps, membranes were incubated with a goat anti-mouse horseradish peroxidase- (HRP-) conjugated secondary antibody (Santa Cruz) and developed with ECL reagents. Bands were quantitated by densitometry and the optical density (OD) of the TCRζ band normalized against the CD3ε band (used as a load control protein) for each sample.
9
Prostate Acid Phosphatase Protein Expression
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The testicular tissue was lysed with a RIPA buffer with protease inhibitors. The total soluble protein was quantified by the BCA protein assay. The total protein (30 μg) was loaded onto a 10% SDS-PAGE gel, separated by electrophoresis, and transferred onto a polyvinylidene difluoride membrane. The blots were blocked with 5% skim milk and incubated with a primary antibody overnight at 4 °C, followed by incubation with a secondary antibody for 1 h at room temperature, and measured with an Infrared Imaging System (LI-COR, Lincoln, NE, USA). The protein expression was normalized by the detection of β-actin (Abcam, Cambridge, UK). The protein from the testicular tissue was isolated to measure the expression level of PAP, and was probed using the primary monoclonal antibody (Santa Cruz, CA, USA).
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