MVSCs were plated onto glass coverslips, placed in 6-well culture plates and returned to the incubator for a minimum of 24 h to allow for cell attachment and spreading. Cells were fixed and permeabilised by the addition of ice-cold acetone for 1 min. The cells were then washed three times with PBS containing 0.5 % TWEEN-20 (PBS-T), pH 7.5, and incubated for 10 min in blocking solution (5 % non-fat dry milk in PBS-T). Cells were stained for 30 min at room temperature with specific antibodies followed by incubation with an Alexa-488-labelled secondary antibody (Molecular Probes, Eugene, Ore., USA). The cells on coverslips were mounted on slides with antifade medium (Dako). Slide preparations were observed by using a Zeiss Axio Observer.Z1 equipped with a Zeiss 710 and ConfoCor3 laser scanning confocal head (Carl Zeiss). Images were analysed by using Zen 2008 software as we have previously described (McEntee et al. 2011 (link)).
Alexa 488 labelled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-labelled secondary antibody is a fluorescently-conjugated antibody used for detection and visualization in various immunoassays and microscopy techniques. It binds to primary antibodies and emits green fluorescence upon excitation, allowing for the identification and localization of target proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti fade medium, by Agilent Technologies (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Confocor 3, by Zeiss (1 mentions)
Cox 2, by Abcam (1 mentions)
Mpges 1, by Abcam (1 mentions)
Normal donkey serum (nds), by Merck Group (1 mentions)
Nunc lab tek 8 well coverslips, by Thermo Fisher Scientific (1 mentions)
Zen microscope software, by Zeiss (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Z1 axioimager, by Zeiss (1 mentions)
6 protocols using alexa 488 labelled secondary antibody
1
Immunofluorescence Staining of Adherent Cells
2
Drosophila Embryo Antibody Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryos aged 0–16 hours AEL were collected from the UbxCPTI000601 or hthCPTI000378 lines at 25°C. Embryos were washed with tap water and dechorionated in a solution of commercial bleach at room temperature (RT). Embryos were washed with water and fixed with 4% formaldehyde for 30 minutes at RT. Fixed embryos were washed twice in PTX (PTX; PBS, 0.1% Triton X-100) and once with PBTX (PBTX; PBS, 0.1% BSA, 0.1% Triton X-100). After washing, embryos were incubated in PBTX rolling for 2 hours at 4°C to block nonspecific protein binding sites. PBTX was replaced with a primary α-GFP antibody (Molecular Probes) diluted in PBTX and incubated overnight at 4°C. The primary antibody was removed and the embryos were washed 3 times with PBTX and incubated for 1 hour at 4°C with rolling. Alexa 488 labelled secondary antibody (Molecular Probes) was added and incubated for 1 hour 30 minutes at RT. The embryos were then washed 3 times with PBTX over a 1-hour period at RT. After removing the excess PBTX, the embryos were mounted in Citifluor and visualized using a Zeiss Axiophot fluorescence microscope.
To double-label the embryos, the same procedure as described above was performed except with the use of two primary antibodies followed by subsequent incubation with two species-specific secondary antibodies.
To double-label the embryos, the same procedure as described above was performed except with the use of two primary antibodies followed by subsequent incubation with two species-specific secondary antibodies.
3
Immunofluorescence Staining of Adherent Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A7r5 and A10 cells were plated onto glass coverslips, placed in six well culture plates and returned to the incubator for a minimum of 24 h to allow for cell attachment and spreading. Cells were fixed and permeabilised by the addition of ice-cold acetone for 1 min. The cells were then washed multiple times (3X) with phosphate-buffered saline (PBS) containing 0.5% TWEEN-20 (PBS-T); pH 7.5, and incubated for 10 min in blocking solution (5% non-fat dry milk in PBS-T). Cells were stained for 30 min at room temperature with specific antibodies followed by incubation with an Alexa 488-labelled secondary antibody (Molecular Probes, Eugene, OR, USA). The cells on cover slips were mounted on slides with antifade medium (Dako). Slide preparations were observed using a Zeiss Axio Observer. Z1 equipped with a Zeiss 710 and ConfoCor3 laser scanning confocal head (Carl Zeiss, Inc.). Images were analyzed using Zen 2008 software as previously described [24 (link)].
4
Immunofluorescence Analysis of COX-2 and mPGES-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells at different confluencies were washed in PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. For permeabilization, the cells were incubated with 0.05% Triton X-100 solution at room temperature for 10 min and blocked with 5% normal goat serum (NGS) at room temperature for 1 hour. Then, the cells were stained with specific primary antibodies against COX-2 and mPGES-1 (Abcam, Cambridge, MA, USA) followed by 2 hours of incubation with Alexa 488-labelled secondary antibody (1:1,000; Molecular Probes, Eugene, OR, USA). The nuclei were stained with DAPI. The images were captured by a confocal microscope.
5
Visualizing RRM2 Expression in Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U-87MG tumor and endometriotic primary cells were seeded on Nunc Lab-Tek 8-well coverslips (Thermo Fisher Scientific, Waltham, MA, USA) and when they had become about 50% confluent, cells were transfected with CPP/siRNA NPs. The cells were fixed with 4% formaldehyde (Naxo, Tartu, Estonia) 48 h post-transfection. Nonspecific staining was reduced using species-specific blocking solution containing 0.25% TritonX-100 in PBS supplemented with 5% normal donkey serum (Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated overnight with primary R2 antibody (#sc-10846, Santa Cruz, Dallas, TX, USA) against RRM2 in blocking solution (1:500), followed by 3 h incubation with Alexa488-labelled secondary antibody (#A-11055, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in blocking solution (1:1000). Nuclei were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Cells were visualized using an LSM710 confocal microscope and images were analyzed using ZEN microscope software (Zeiss, Oberkochen, Germany).
6
Multimodal Analysis of Cardiac Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded cardiac tissue sections were incubated with anti-arginase 1 (clone N-20, 1:100, Santa Cruz, USA) primary antibody followed by incubation with secondary antibody biotinylated rabbit anti-goat (1: 400, Dianova, Germany). Biotin was detected with alkaline phosphatase-labelled streptavidin (Agilent, USA) and visualized using RED (Agilent, USA) as a chromogen. Proteins and enzymes were inactivated with heat and alkaline pH prior to incubation with anti-CD68 (1:250, Amsbio #1518), followed by incubation with Alexa488-labelled secondary antibody (1:400, donkey anti-rabbit, Invitrogen, Germany). DAPI (Sigma, Germany) was used to stain nuclei and sections mounted with Fluoromount-G (Southern Biotech, USA). Negative controls were performed by omitting the primary antibodies. Images were acquired with an AxioImager Z1 (Zeiss MicroImaging GmbH, Germany). All evaluations were performed in a blinded manner.
5 µm paraffin-sections of rat LV myocardium were stained with picrosirius red to obtain collagen content (52 (link)).
5 µm paraffin-sections of rat LV myocardium were stained with picrosirius red to obtain collagen content (52 (link)).
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!