Intracellular immunofluorescence staining of the intracellular signaling molecules in the immunological synapse of activated B cells was performed according to our published protocols (41 (link)). Briefly, B cells were fixed with 4% PFA for 30 min at room temperature. After washing with 1× phosphate-buffered saline (PBS), B cells were permeabilized with 0.2% Triton X-100 for 20 min and then blocked with goat nonspecific IgG (100 μg/ml; 005-000-003, Jackson ImmunoResearch Laboratory) for 1 hour at room temperature. Subsequently, cells were incubated with various primary antibodies including anti–phospho-Syk (pY525/526) antibody (1:500 dilution; #2711, Cell Signaling Technology), anti–phospho-PI3K p85 (pY458)/p55 (pY199) antibody (1:500 dilution; #4228, Cell Signaling Technology), and anti–phospho-BLNK antibody (1:500 dilution; BioDragon) at 37°C for 1 hour, followed by Alexa Fluor 568–conjugated F(ab′)2 fragment goat anti-rabbit IgG (1:2000 dilution; A21069, Invitrogen) as the secondary antibody. TIRFM images were analyzed using ImageJ (NIH).
Alexa fluor 568 conjugated f ab 2 fragment goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Alexa Fluor 568–conjugated F(ab′)2 fragment goat anti-rabbit IgG is a secondary antibody reagent. It is composed of F(ab′)2 fragments of goat anti-rabbit IgG that are conjugated to the Alexa Fluor 568 fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using alexa fluor 568 conjugated f ab 2 fragment goat anti rabbit igg
1
Intracellular Immunofluorescence Staining of Activated B Cells
2
Intracellular Signaling Molecules in Activated B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular immunofluorescence staining of the signalling molecule in the IS of activated B cells were performed according to our published protocols7 (link)15 (link)32 (link). Briefly, B cells were fixed with 4% PFA fixation for 30 min at RT. After washed with 1 × PBS, B cells were permeabilized with 0.2% Triton X-100 for 20 min and then blocked with 100 μg ml−1 goat non-specific IgG (Jackson ImmunoResearch Laboratory, 005-000-003) for 1 h at RT. Subsequently, cells were incubated with various primary antibodies including Anti-phospho-Syk (pY525/526) Ab (Cell Signaling, #2711, 1:500 dilution), Anti-phospho-PI3K p85 (pY458)/p55 (pY199) Ab (Cell Signaling, #4228, 1:500 dilution) and Anti-phospho-BLNK Ab (Biodragon, 1:500 dilution) at 37 °C for 1 h and then Alexa Fluor 568 conjugated F(ab)2 fragment goat anti- rabbit IgG (Invitrogen, 1:2,000 dilution) was used as the secondary antibody. TIRFM Images were processed with Image J (NIH, USA)7 (link)8 (link)15 (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!