The immunohistochemical staining used to detect c-Fos activity was performed using the free-floating section technique. The sections were washed three times in PBS (pH 7.4), immersed in a 3% solution of H2O2 in PBS for 5 min, washed three times in PBS, incubated in BSA (Capricorn scientific, cat no. BSA-1S) for 1 h at 22±1˚C and then incubated with a Rabbit polyclonal antibody (anti-c-Fos, hippocampus 1:400, prefrontal cortex 1:500, Abcam, cat no. Ab190289) for 15 h at 4˚C. The sections were washed three times in PBS, and then the sections were incubated with goat anti-rabbit IgG H&L (HRP) (1:500, Abcam, cat no. Ab205718) for 1 h at 22±1˚C. Subsequently, they were washed three times in PBS, and next incubated with DAB (Abcam, cat no. Ab64238) for 5 min and then they were washed three times in PBS. Finally, the sections were mounted on slides, air dried, dehydrated in ethanol solutions and xylene, and a cover slip placed on top with Mounting Medium (Thermo Fisher Scientific, Inc., cat no. SP15-500). c-Fos was detected using dark-brown staining. The number of c-Fos positive cells were counted in three regions of the dorsal hippocampus (CA1, CA3, and Dentate gyrus; DG) and the medial prefrontal cortex (mPFC). Images of the two sections per animal brains were counted using ImageJ (Version 1.53; National Institutes of Health).
Bovine serum albumin (bsa)
Manufactured by Capricorn
Sourced in United States
The BSA (Bovine Serum Albumin) is a laboratory product used as a protein standard. It is a widely used reference material for various analytical and experimental purposes in life science research.
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Lab products found in correlation
Triton x 100, by Bio Basic (2 mentions)
Xponent 4, by DiaSorin (2 mentions)
Tween 20, by Promega (2 mentions)
Flexmap 3d, by DiaSorin (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Anti lc3b antibody, by Thermo Fisher Scientific (1 mentions)
Permount, by Merck Group (1 mentions)
Mounting medium, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Leica confocal software, by Leica camera (1 mentions)
D9542, by Merck Group (1 mentions)
Minimum essential medium eagle, by Merck Group (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Bio Basic (1 mentions)
Eclipse ts2, by Nikon (1 mentions)
Triton x 100, by Avantor (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Bio plex manager 6, by Bio-Rad (1 mentions)
Convenience 36 plex mouse procartaplex panel 1a, by Thermo Fisher Scientific (1 mentions)
9 protocols using bovine serum albumin (bsa)
1
Immunohistochemistry for c-Fos Detection
2
FFPE Liver Immunostaining for LC3B
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After deparaffinization and rehydration of FFPE liver sections according to a standard protocol, antigen retrieval was conducted by boiling in 10 mM sodium citrate buffer. After cooling down, the slides were permaebilized and blocked using 0.4% Triton X-100 (BioBasic) and 5% BSA (Capricorn) in PBS, consecutively. After that, the slides were incubated with an anti-LC3B antibody (Thermo Fisher Scientific) overnight at 4 °C. After twice washing with PBS (10 minutes per wash), the slides were further incubated with a Dylight 488 conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific) for 2 hours in the dark at room temperature. Finally, the slides were washed and mounted by using Permount (Merck) for further analysis under fluorescence microscope.
3
Multiplex Cytokine Profiling of Cell Supernatants
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Supernatants were collected for Luminex analysis using the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Customized 7-plex Panel (Merck Millipore) to measure the following targets: IL-1α, IL-1β, IL-1RA, IL-6, IL-8, IL-10, and TNF-α. Harvested supernatants and standards were incubated with fluorescent-coded magnetic beads pre-coated with respective antibodies in a black 96-well clear-bottom plate overnight at 4°C. After incubation, plates were washed five times with wash buffer (PBS with 1% BSA (Capricorn Scientific) and 0.05% Tween-20 (Promega)). Sample-antibody-bead complexes were incubated with biotinylated detection antibodies for 1-h, and subsequently, Streptavidin-PE was added and incubated for another 30 min. Plates were washed five times again, before sample-antibody-bead complexes were re-suspended in sheath fluid for acquisition on the FLEXMAP® 3D (Luminex) using xPONENT® 4.0 (Luminex) software. Data analysis was done on Bio-Plex ManagerTM 6.1.1 (Bio-Rad). Standard curves were generated with a five-parameter logistic algorithm, reporting values for both mean florescence intensity (MFI) and concentration data.
4
Oleic Acid Uptake in p63-Silenced Cells
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After silencing total p63 and TAp63α in THLE2 cells, cell culture medium was supplemented with 1 mM of oleic acid (Sigma Aldrich, USA) bound to fatty-acid-free bovine serum albumin (BSA) (Capricorn) (2:1 molar ratio). Controls were supplemented with BSA alone. THLE2 cells were incubated with oleic acid and BSA during 12 h. After treatments, the coverlips were placed in a 24-well plate with 500 μl of Minimum Essential Medium Eagle (Sigma-Aldrich, USA) complete medium and incubated (37 °C) during 40 min with BODIPY (green) 493/503 (1:200 dilution) (Thero Fisher, USA). After incubation, coverlips were washed with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde during 10 min. The coverslips were mounted in aqueous medium (FluoroGel #17985-10) with DAPI (D-9,542, Sigma-Aldrich, USA) (blue) (1:1,000) for preserving cell fluorescence. Confocal images were collected using a Leica confocal microscope (Leica A0B5-SPSX) equipped with a high grade colour corrected plan apochromat lens for confocal scanning × 63/1.32 objective. Leica Confocal Software was used for acquisition and analysis. Images are combinations of optical sections taken in the z axis at 0.5-μm intervals.
5
Trypsin Enzyme Activity Assay
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Two methods for incubation of samples with trypsin having differences in reaction buffers, incubation period and temperature were employed. BSA (Capricorn Scientific GmbH, fraction V, origin USA, Ebsdorfergrund, Germany) was prepared as 4mg/ml stock in distilled water and used as substrate for the activity of trypsin enzyme in both methods.
6
Immunofluorescence Identification of Liver Capillaries
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Immunofluorescence of CD34 is a method used to identify capillaries and sinusoids in liver using anti-CD34 monoclonal mouse antibody (CD34) (US Biological). In a typical procedure, the liver sections (5 μm) were deparaffinized and rehydrated according to standard protocol. Next, antigen retrieval was performed by boiling the sectioned slides in 10 mM sodium citrate buffer (pH 6.0). After cooling down to room temperature and washed with DI water, the sections were permeabilized with a permeabilization solution containing 1% BSA (Capricorn), 0.4% Triton X-100 (Bio Basic) in PBS for 10 minutes and were further incubated with a blocking solution (5% BSA) for 30 minutes. Afterward, the permeabilized sections were incubated with an anti-CD34 monoclonal mouse antibody (FITC labeling) for 24 hours at 4 °C. After washing twice with 1% BSA in PBS, the stained sections were mounted with Permount. The CD34+ capillaries and sinusoids in liver were then observed under an inverted fluorescence microscope (Nikon, Eclipse Ts2).
7
Visualizing Actin Polymerization in T Cells
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To visualise actin polymerisation in Jurkat T cells and N1KO cells, 2 × 105 cells in 100 μL starvation medium were treated with 10 nM AX-024 or left untreated. Cells were incubated at 37 °C for 30 min, followed by stimulation with 5 μg/mL anti-CD3ε antibody (clone OKT3) at 37 °C for 5 min. The cells were then fixed with 4% paraformaldehyde (Sigma-Aldrich) and rendered permeable with 0.1% Triton X-100 (Amresco, NY, USA). After washing with 1 × PBS, samples were blocked with 1% bovine serum albumin (Capricorn, CA, USA) and washed with 1 × PBS. Cells were then stained with rhodamine-phalloidin-conjugated FITC (Abcam, MA, USA) at a dilution of 1:200 and incubated at room temperature for 20 min [46 (link)]. Finally, cells were seeded into a Tomodish at a total volume of approximately 100 μL of the total volume and observed for green fluorescence signals under the holotomographic microscope (Tomocube). ImageJ software was used to measure the value of integrated density, area of selected cell, and mean fluorescence of background before calculating the corrected total fluorescence (CTCF) intensity using the following formula: CTCF = integrated density – (area of selected cell × mean fluorescence of background readings).
8
Multiplexed Cytokine and Chemokine Profiling
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Serum cytokine and chemokine levels were measured with Cytokine & Chemokine Convenience 36-Plex Mouse ProcartaPlex Panel 1A (Thermo Fisher Scientific) according to the manufacturer’s protocol. Plasma was incubated with fluorescent coded magnetic beads precoated with respective antibodies in a black 96-well clear-bottom plate overnight at 4°C. After incubation, plates were washed five times with wash buffer [PBS with 1% bovine serum albumin (Capricorn Scientific) and 0.05% Tween-20 (Promega)]. Sample antibody–bead complexes were incubated with biotinylated detection antibodies for 1 hour and washed five times with wash buffer. Subsequently, streptavidin–phycoerythrin (PE) was added and incubated for another 30 min. Plates were washed five times again, before sample antibody–bead complexes were resuspended in sheath fluid for acquisition on the FLEXMAP 3D (Luminex) using xPONENT 4.0 (Luminex) software. Data analysis was performed on Bio-Plex Manager 6.1.1 (Bio-Rad). Standard curves were generated with a 5-PL (5-parameter logistic) algorithm, reporting values for both MFI and concentration data.
9
Immunofluorescence Assay for Bcl-2
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Cells were treated with IC 50 /2 doses of drugs for 24 hr. Fixed HepG2 cells were blocked with bovine serum albumin (Capricorn, Ebsdorfergrund, Germany) and incubated with anti-human Bcl-2 antibody (1:100 dilution, Biocare med) overnight at 4 °C. Next day, the cells were rinsed with PBS three times and incubated with goat anti-mouse IgG DyLight 488 (1:200, Thermo Scientific, Waltham, MA, USA) for 90 min at 37 °C. After rinsing three times with PBS, the cells were counterstained with Hoechst 33258 (Thermo Scientific) for labeling the nuclei. Images were taken with a laser scanning confocal microscope (LSM 700, Zeiss).
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