Cells were two times washed with PBS before being fixed with 2% PFA at RT for 30 min. After another two washes with PBS, cells were permeabilized with 0.5% Triton in PBS for 15 min. Cells were washed again 3× in PBS and blocked with blocking buffer (5% donkey or goat serum in PBS) for 10 min. Cells were incubated with primary antibody (1:100 in blocking buffer) overnight at 4 °C. The next day, cells were washed with PBS, PBS + 0.05% Tween-20 and again with PBS before incubation with secondary antibody (1:500) in DAPI (1:5000) and 2% Human Serum for 60 min at RT in the dark. Cells were washed with PBS, PBS + 0.05% Tween-20 and PBS and stored in PBS at 4 °C until analysis. The following primary antibodies were used: mouse anti-human CD31 (M0823, Dako, Santa Clara, CA, USA), rabbit anti-human SM22α (ab14106, Abcam, Cambridge, MA, USA). Alexa Fluor 647 donkey anti-mouse (A31571, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568 goat anti-rabbit (A21069, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. The cells were analyzed using the EVOS FL cell imaging system (Life Technologies, Carlsbad, CA, USA).
Alexa fluor 568 goat
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 568 goat is a fluorescent dye conjugate used in various biological applications. It is a bright, photostable dye that can be excited using the 568 nm laser line. The dye is covalently attached to goat immunoglobulins, making it suitable for labeling and detection of target proteins or molecules in immunoassays and microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Ab14106, by Abcam (1 mentions)
A21069, by Thermo Fisher Scientific (1 mentions)
M0823, by Agilent Technologies (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
A31571, by Thermo Fisher Scientific (1 mentions)
Operetta cls high content analysis system, by PerkinElmer (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Cleaved casp3, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
Mouse anti nc82, by Developmental Studies Hybridoma Bank (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Anti pericentrin, by Abcam (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Lab tek chamber slide, by Thermo Fisher Scientific (1 mentions)
5 protocols using alexa fluor 568 goat
1
Immunofluorescence Staining of Endothelial and Smooth Muscle Cells
2
High-Content Analysis of DNA Damage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded in Corning black flat-bottom 96-well plates 24 h before drug treatment. After treatment, cells were fixed with 4% paraformaldehyde for 10 min, permeabilised with 0.1% Triton X-100, 0.1% Tween-20, and 1×PBS (PBSTT) for 10 min at room temperature and blocked for 30 min with BS (same as flow). Primary antibody in BS was added for 1 h at room temperature (mouse anti-γH2AX S139, Millipore #05-636, 1:2000 and rabbit anti-phospho-RPA32 S4/8, Bethyl#A300-245A, 1:1000). After washing, secondary antibody (Alexa Fluor 488 goat anti-rabbit, Invitrogen#A11034, 1:500 and Alexa Fluor 568 goat anti-mouse, Invitrogen#A11019, 1:500) plus Hoechst 33342 (Invitrogen#H3570, 1:10,000) in BS was added for 1 h at room temperature. After washing, images were acquired using the Operetta CLS High-Content Analysis System (Perkin Elmer), and analysis was performed using the Harmony 4.5 software.
3
Immunofluorescence Analysis of 3D Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were plated in 3D cultures as described above in 24-well plates with or without HCQ. Well coatings containing the cells were smeared on permafrost slides and fixed for 10 min with 4% PFA. The cells were washed 10 min with PBS and permeabilized with PBS containing 0.5%Triton X-100 (Sigma) for 10 min, at 4 °C. Permeabilized cells were washed with PBS (3 × 15 min) and blocked with 10% goat serum for 1 h and incubated overnight at 4 °C with primary antibody. Slides were incubated with anti-LAMP1 from Novus Biologicals (NBP2-25154) and from Cell Signaling Technology anti-LC3 (CST, 12741), Ki67 (CST 9449) or cleaved CASP3 (CST, 9661) in 5% BSA and Tris-buffered saline and 0.05% Tween 20 (TBS-T) at 4 °C overnight. Later, the slides were incubated with either Alexa fluor 488 goat anti-rabbit and Alexa fluor 568 goat anti-mouse secondary antibodies or Alexa fluor 488 goat anti-mouse and Alexa fluor 594 goat anti-rabbit (Invitrogen) for 1 h, mounted with Vectashield mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) and imaged using a Zeiss LSM 780 confocal microscope.
4
Immunohistochemical Analysis of Drosophila Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dissection and immunohistochemistry of fly brains were performed as previously described with minor modifications (Jenett et al., 2012 (link)). Brains of 3–10 day old female flies were dissected in Schneider's Insect medium and fixed in 2% paraformaldehyde in Schneider's medium for 50 min at room temperature (RT). After washing in PBT (0.5% Triton X-100 in PBS), brains were blocked in 3% normal goat serum (or normal donkey serum, depending on the secondary antibody) for 90 min. Brains were then incubated in primary antibodies diluted in PBT for 2–4 days on a nutator at 4°C, washed three times in PBT for 30 min or longer, then incubated in secondary antibodies diluted in PBT for 2–4 days on a nutator at 4°C. Brains were washed thoroughly in PBT four times for 30 min or longer, and mounted in Vectashield (Vector laboratories, CA) for imaging. The following antibodies were used: anti-GFP (1:1000; Invitrogen; #AB124), mouse anti-nc82 (1:50; Developmental Studies Hybridoma Bank, Univ. Iowa), and cross-adsorbed secondary antibodies to IgG (H+L): goat Alexa Fluor 488 anti-rabbit (1:800; Invitrogen A11034) and goat Alexa Fluor 568 (1:400; Invitrogen A11031).
5
Immunofluorescence Microscopy of PANC-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PANC-1 cells were cultured in a Lab-Tek chamber slide (Nalge Nunc International, Rochester, NY, USA) at a density of 20,000 cells/well. After 48 h, cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min. After being permeabilized with 0.5% Triton X-100 in PBS for 10 min, the cells were blocked with 1% BSA in PBS and incubated with primary antibodies overnight at 4 °C. Primary antibodies used in these studies were anti-pericentrin (Abcam, Cambridge, UK), anti-α-tubulin (Abcam), human anti-CREST (Immuno Vision Inc., Springdale, AR, USA) and anti-cleaved caspase-3 (1:400, Cell Signaling Technology). The cells were then washed three times with PBS, and incubated with the indicated secondary antibody for 2 h at 25 °C. Secondary antibodies were goat Alexa Fluor 568 (Invitrogen), goat Alexa Fluor 488 (Abcam), and goat anti-Human IgG-FITC (Invitrogen). Nuclei were counterstained with DAPI and mounted with ProLong Gold Antifade (Invitrogen). Images were captured using a ZEISS LSM 710 confocal microscope and processed using ZEN software (ZEISS International, Oberkochen, DE).
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!