HeLa cells and human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 U ml−1) and streptomycin (100 μg ml−1) at 37°C, 5% CO2. For microscopy HeLa cells were grown to 50–70% confluence on 18 × 18 glass coverslips and transfected with expression constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 24 hours post-transfection, cells were fixed with 4% paraformaldehyde (15 min at room temperature) and permeabilised with 1% Triton-X-100 in phosphate buffered saline (PBS). JMJD6 (ab10526, Abcam), anti-BrdU (B8434, Sigma), anti-HA (H6908, Sigma) and anti-U2AF65 (ab37483, Abcam) were used as primary antibodies, Cy3-coupled anti-mouse (Jackson Immuno Research), Alexa647 anti-mouse (Invitrogen) and Alexa488 anti-rabbit (Invitrogen) were used as secondary antibodies.
Alexa 647 anti mouse
Manufactured by Thermo Fisher Scientific
Alexa Fluor 647 anti-mouse is a fluorescent antibody used for detection and quantification of mouse proteins in various immunoassays and imaging applications. It consists of a mouse-specific antibody conjugated to the Alexa Fluor 647 dye, which has excitation and emission maxima of 650 nm and 665 nm, respectively.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Lsm 700 confocal microscope, by Zeiss (4 mentions)
Chicken anti gfp, by Thermo Fisher Scientific (2 mentions)
Alexa 488 anti chicken, by Thermo Fisher Scientific (2 mentions)
Rat anti flag, by Novus Biologicals (2 mentions)
Ss8x9 secureseal spacers, by Thermo Fisher Scientific (2 mentions)
Rabbit anti th, by Merck Group (2 mentions)
Rat anti cd8, by Thermo Fisher Scientific (2 mentions)
Alexa 488 anti rat, by Thermo Fisher Scientific (2 mentions)
Slowfade gold, by Thermo Fisher Scientific (2 mentions)
Rabbit anti ha, by Cell Signaling Technology (2 mentions)
Anti mouse cy3, by Jackson ImmunoResearch (2 mentions)
Mouse anti nc82, by Developmental Studies Hybridoma Bank (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Ab10526, by Abcam (1 mentions)
Ab37483, by Abcam (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
B8434, by Merck Group (1 mentions)
Hcs cellmask red stain, by Thermo Fisher Scientific (1 mentions)
4 protocols using alexa 647 anti mouse
1
Immunofluorescence Microscopy of HeLa and HEK293T
2
Antibody Labeling and Cellular Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All conjugated and unconjugated primary antibodies used in this study are listed in Table 2 . Indirect immunofluorescence was performed using secondary antibodies conjugated with Alexa-647 anti-Mouse (Invitrogen, Cat. A-21236), Alexa-555 anti-Rat (Invitrogen, Cat. A-21434) and Alexa-488 anti-Rabbit (Invitrogen, Cat. A-11034). 10 mg/ml Hoechst 33342 stock solution was purchased from Life Technologies (Cat. H3570). 20xPBS was purchased from Santa Cruz Biotechnology (Cat. SC-362299). 30% hydrogen peroxide solution was purchased from Sigma-Aldrich (Cat. 216763). PBS-based Odyssey blocking buffer was purchased from LI-COR (Cat. 927–40150). All reagents for the Leica BOND RX were purchased from Leica Microsystems. HCS CellMask Red Stain and Mito-tracker Green stains were purchased from ThermoFischer (catalog numbers H32712, R37112 and M751, respectively).
3
Whole-Mount Immunostaining of Fly Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunostaining of whole-mount brains or VNCs was performed as follows: brains or VNCs of 3- to 7-day-old flies were dissected in PBS, fixed in 4% paraformaldehyde (PFA) for 20 min, and subjected to chicken anti-GFP (1:1000, In-vitrogen), rabbit anti-TH (1:1000, Millipore), rat anti-CD8 (1:200, Invitrogen), mouse anti-nc82 (1:25, Developmental Studies Hybridoma Bank), or all four at 4°C for 18–40 hr, followed by incubation with fluorescent Alexa 488 anti-chicken (1:1000, Invitrogen), Alexa 488 anti-rat (1:200, Invitrogen), Alexa 568 anti-rabbit (1:1000, Invitrogen), Alexa 647 anti-mouse (1:1000, Invitrogen), or Cy3 anti-mouse (1:200, Jackson Immunoresearch) secondary antibodies for ~16 hr at 4°C. Brains or VNCs were mounted in Vectashield (Vector Laboratories) or SlowFade Gold using SS8X9-SecureSeal spacers (both Thermo Fisher Scientific). Images were obtained on an LSM510 or LSM700 confocal microscope (Zeiss) with 1-μm- or 3-μm-thick sections under 10x or 25x magnification. MultiColor FlpOut (MCFO) analysis of select driver combinations was performed as described previously (Nern et al., 2015 (link)) using rat anti-FLAG (1:200, Novus Biologicals) and rabbit anti-HA (1:300, Cell Signaling Technology) antibodies.
4
Whole-Mount Immunostaining of Fly Brains
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!