Cells infected with BAV304a, mutant BAdV-3s, or transfected with plasmid DNAs were processed as described earlier (Wodrich et al., 2006 (link)). Finally, cells were mounted by mounting buffer (Vector Laboratories Inc.) with DAPI and imaged under confocal microscope TCS SP5 (Leica).
Mounting buffer
Manufactured by Vector Laboratories
Sourced in United States
Mounting buffer is a solution used to prepare specimens for microscopic analysis. It is designed to aid in the mounting and preservation of tissue samples or other biological materials on microscope slides. The buffer provides a suitable environment to maintain the structural integrity and clarity of the specimen during observation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5 confocal spectral microscope imaging system, by Leica (7 mentions)
Tcs sp5, by Leica (1 mentions)
Ultra 5 block, by Thermo Fisher Scientific (1 mentions)
Axiocam camera system, by Zeiss (1 mentions)
Axioskop photomicroscope, by Zeiss (1 mentions)
Evos fl auto 2 imaging system, by Thermo Fisher Scientific (1 mentions)
Argon krypton laser, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
11 protocols using mounting buffer
1
Imaging of Virus-Infected and Transfected Cells
2
Immunofluorescence Staining of RAW 264.7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW 264.7 cells (5 × 104 cells per well) were cultured on cover slips in 6-well plates and treated with 0.1% DMSO or 20 μM TQ-6 with or without LPS stimulation for 30 min. The cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After incubation, the cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% BSA for 30 min. The cells were incubated with primary antibodies overnight at 4 °C, subsequently washed 3 times with PBS, and incubated with secondary antibodies for 1 h at room temperature. The samples were stained with 4′,6-diamidino-2-phenylindole (DAPI, 30 μM) and mounted using a mounting buffer (Vector Laboratories) on a glass slice. The samples were detected under a Leica TCS SP5 confocal spectral microscope imaging system using an argon or krypton laser (Mannheim, Germany) [15 (link)].
3
Placental Cell Phenotyping via Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the phenotype of H3K9ac expressing cells in the placenta tissue, double immunofluorescence staining was performed. Placenta tissue of both the control and the GDM group were stained, using CK7 as a marker for EVTs and CD31 as a marker for foetal endothelial cells.
Tissue samples from the same patient collective were used for double immunofluorescence staining as for immunohistochemistry. Pre-treatment of the slides (deparaffinising, blocking of endogenous peroxidase activity, de-masking of protein epitopes) was identical to that used for immunohistochemistry. Blocking solution (Ultra V–Block, Thermo Scientific, Lab Vision, Fremont, CA, USA) was applied for 15 min in order to prevent any unspecific antigen-antibody binding. Thereafter, the primary antibodies were mixed and applied together (seeTable 2 ). Following this, the slides were incubated with the secondary antibodies (see Table 2 ) for 30 min. The slides were then cover-slipped with minimal light exposure using mounting buffer (Vector Laboratories, Burlingame, CA, USA), which contains DAPI for nuclear counterstaining. The phenotypes were analysed using the fluorescent Axioskop photomicroscope (Zeiss, Oberkochen, Germany). Images were taken with a digital Axiocam camera system (Zeiss, Oberkochen, German).
Tissue samples from the same patient collective were used for double immunofluorescence staining as for immunohistochemistry. Pre-treatment of the slides (deparaffinising, blocking of endogenous peroxidase activity, de-masking of protein epitopes) was identical to that used for immunohistochemistry. Blocking solution (Ultra V–Block, Thermo Scientific, Lab Vision, Fremont, CA, USA) was applied for 15 min in order to prevent any unspecific antigen-antibody binding. Thereafter, the primary antibodies were mixed and applied together (see
4
Immunohistochemical Analysis of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animals of each group were euthanized and perfused with 4% paraformaldehyde in PB. Brains were post-fixed with 4% paraformaldehyde in PB overnight. Before sectioning, the brains were immersed in 30% sucrose in PB for cryoprotection. The brains were sliced in the coronal plane at 50 µm and permeabilized with PB containing 0.2% Triton X-100 and 6% donkey serum for 60 min. These slices were incubated overnight at 4 °C with primary antibodies including anti-caspase-3, anti-NeuN, and anti-Iba-1 antibodies. Subsequently, samples were washed three times with 0.2% Tween 20 in PB and then exposed to secondary antibodies overnight. The prepared slices were then counterstained with DAPI (30 mM) and mounted with mounting buffer (Vector Laboratories, Burlingame, CA, USA) on a glass coverslip. Samples were detected under an Evos FL Auto 2 imaging system using the LED fluorescence light cubes (Thermo Fisher Scientific, Waltham, MA, USA).
5
Immunofluorescence Analysis of Rutaecarpine Effects on LPS-Stimulated RAW 264.7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW 264.7 cells (5 × 104 cells per well) were cultured on cover slips in 6-well plates and treated with 0.1% DMSO or 20 μM rutaecarpine with or without LPS stimulation for 30 min. The cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After incubation, the cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% BSA for 30 min. The cells were incubated with primary antibodies overnight at 4 °C, subsequently washed 3 times with PBS, and incubated with secondary antibodies for 1 h at room temperature. The samples were stained with 4,6-diamidino-2-phenylindole (DAPI, 30 μM) and mounted using a mounting buffer (Vector Laboratories) on a glass slice. The samples were detected under a Leica TCS SP5 confocal spectral microscope imaging system using an argonor krypton laser (Mannheim, Germany).
6
Immunofluorescence Visualization of Cellular Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In 6-well plates, cells at a density of 5 × 104 per well were cultured on cover slips and treated with 0.1% DMSO or 20 μM esculetin with or without LTA stimulation for 1 h. The cover slips were washed with PBS and then fixed with 4% paraformaldehyde at room temperature for 10 min. Cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% BSA for 30 min. After the incubation, cells were titrated with the respective primary antibody overnight at 4 °C, subsequently washed with PBS three times, and titrated with secondary antibody at room temperature for 1 h. The samples were stained with 4,6-diamidino-2-phenylindole (DAPI, 30 μM) and mounted using a mounting buffer (Vector Laboratories) on a glass slice. The observations were spotted under a Leica TCS SP5 confocal spectral microscope imaging system using an argon or krypton laser (Mannheim, Germany).
7
Indirect Immunofluorescence Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Indirect immunofluorescence microscopy was performed as described previously (61 (link)). Cover glass was mounted using mounting buffer (Vector Laboratories) and observed under Nikon Eclipse fluorescence microscope.
8
Immunohistochemical Analysis of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animals of each group were euthanized and perfused with 4% paraformaldehyde in phosphate buffer (PB). The brains were post-fixed with 4% paraformaldehyde in PB overnight. Before sectioning, the brains were immersed in 30% sucrose in PB for cryoprotection. The brains were sliced in the coronal plane at 50 mm and permeabilized with PB containing 0.2% Triton X-100 and 6% donkey serum for 60 minutes. These slices were incubated overnight at 4 °C with primary antibodies. Subsequently, the samples were washed 3 times with 0.2% tween 20 in PB and then exposed to secondary antibodies overnight. The prepared slices were then counterstained with DAPI (30 mM) and mounted using a mounting buffer (Vector Laboratories) on a glass coverslip. The samples were detected under a Leica TCS SP5 confocal spectral microscope imaging system using an argon or krypton laser (Mannheim, Germany).
9
Immunofluorescent Analysis of LTA-treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells at a density of 5 × 104 cells per well were cultured on coverslips in 6-well plates and treated with 0.1% DMSO or 20 μM Rut with or without LTA stimulation for 30 min, washed with PBS, and then fixed with 4% paraformaldehyde at room temperature for 10 min. Cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% BSA for 30 min. After incubation, cells were titrated with primary antibodies (1:100) overnight at 4 °C, consequently washed three times with PBS, and titrated with secondary antibodies at room temperature for 1 h. The samples were stained with 4,6-diamidino-2-phenylindole (DAPI, 30 μM) and mounted using a mounting buffer (Vector Laboratories) on a glass slide. The samples were spotted under a Leica TCS SP5 confocal spectral microscope imaging system using an argon or krypton laser (Mannheim, Germany).
10
Confocal Imaging of p65 Translocation in VSMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A confocal microscopic analysis was performed to identify and evaluate the translocation of p65 in VSMCs. Vascular smooth muscle cells (1 × 105 cells/cover slide) were placed on cover slides and allowed to adhere in a cell culture incubator overnight. Vascular smooth muscle cells were treated as the design of the experiment, then fixed with 4% paraformaldehyde for 30 min. and permeabilized with 80% methanol for 15 min. After incubation with 3% skimmed milk in PBS for 60 min., the preparation was incubated for 1 hr with a primary Ab (anti-p65 mouse mAb; 1:80). Cells were then washed three times with PBS and exposed to the secondary Ab [FITC-conjugated antimouse immunoglobin G (IgG; Santa Cruz Biotechnology) at 1:100, 1% BSA/PBS] for 60 min. Slides were counterstained with 0.1 μg/ml DAPI (Sigma-Aldrich) and mounted with mounting buffer (Vector Laboratories, Burlingame, CA, USA) under a glass coverslip on a Leica TCS SP5 Confocal Spectral Microscope Imaging System using an argon/krypton laser (Mannheim, Germany).
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!