The largest database of trusted experimental protocols

4 protocols using alexa fluor 488 goat anti rabbit igg

1

Immunofluorescence Assay for BDNF and VEGF

Check if the same lab product or an alternative is used in the 5 most similar protocols
To perform the immunofluorescence assay, the treated wells were washed with PBS and fixed at RT for 30 minutes in 4% paraformaldehyde. After blocking with 3% normal goat serum and 1% bovine serum albumin (Vector Laboratory, Burlingame, CA, USA) for 1 hour, the well plate was incubated with primary antibodies to BDNF (1:500; Santa Cruz Biotechnology) and VEGF (1:500, Santa Cruz Biotechnology) at 4°C overnight. After plate washes, the plates were incubated with secondary Alexa Fluor 488-goat anti-rabbit IgG (1:400; Vector Laboratory) and Alexa Fluor 594-goat anti-mouse IgG (1:400; Vector Laboratory) antibodies for 1.5 hours at RT. Cells were counterstained with a 4′,6-diamidino-2-phenylindole mounting solution (Vector Laboratory), and slide images were analyzed by fluorescence microscopy.
+ Open protocol
+ Expand
2

Immunofluorescence Analysis of Embryo Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols
Embryo isolation and staging were done as described previously4 (link). The immunofluorescence analysis and AP staining were performed essentially as described previously4 (link). The primary antibodies used were as follows: anti-AP2γ rabbit polyclonal, 1:500 (cat#sc-8977; Santa Cruz Biotechnology); anti-ETMT1/GLP mouse monoclonal, 5µg/mL (cat#PP-B0422-00; R&D Systems); anti-H3K9me2 mouse monoclonal, 1:500 (cat#ab1220; Abcam); anti-SOX2 goat polyclonal, 1:200 (cat#sc-17320, Santa Cruz Biotechnology). The following secondary antibodies from Molecular Probes (Eugene, OR) were used at a dilution of 1:500: Alexa Fluor 488 goat anti-rabbit IgG; Alexa Fluor 568 goat anti-mouse IgG. The stained embryos were mounted with VECTASHIELD Antifade Mounting Medium (cat#H-1000; VECTOR LABORATORIES). The immunofluorescence images and the AP staining images were taken by a confocal microscope (Zeiss LSM880) and a stereomicroscope (Leica M80), respectively. The image analyses were done by using ImageJ/Fiji software.
+ Open protocol
+ Expand
3

Confocal Microscopy of LDLR Localization

Check if the same lab product or an alternative is used in the 5 most similar protocols
Confocal microscopy was carried out as described previously (13 (link), 25 (link), 30 (link)). Briefly, HEK293 cells seeded onto the coverslips (1.0 × 105 cells/ml) were transfected with the WT or mutant LDLR cDNA as indicated. At 48 h later, cells were fixed with 4% paraformaldehyde and permeabilized with cold methanol. After blocking with 1% BSA, the cells were incubated with an anti-LDLR monoclonal Ab and an anti-Na+/K+-ATPase polyclonal Ab (1:200) (Abcam). Ab binding was detected using Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 568 goat anti-mouse IgG. After washing, coverslips were mounted on the slides with Antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Localizations of LDLR and Na+/K+-ATPase in the transfected cells were determined using a Leica SP5 laser-scanning confocal microscope (filters: 461 nm for DAPI, 519 nm for Fluor 488, and 603 nm for Fluor 568).
+ Open protocol
+ Expand
4

Immunofluorescence Analysis of Embryo Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols
Embryo isolation and staging were done as described previously4 (link). The immunofluorescence analysis and AP staining were performed essentially as described previously4 (link). The primary antibodies used were as follows: anti-AP2γ rabbit polyclonal, 1:500 (cat#sc-8977; Santa Cruz Biotechnology); anti-ETMT1/GLP mouse monoclonal, 5µg/mL (cat#PP-B0422-00; R&D Systems); anti-H3K9me2 mouse monoclonal, 1:500 (cat#ab1220; Abcam); anti-SOX2 goat polyclonal, 1:200 (cat#sc-17320, Santa Cruz Biotechnology). The following secondary antibodies from Molecular Probes (Eugene, OR) were used at a dilution of 1:500: Alexa Fluor 488 goat anti-rabbit IgG; Alexa Fluor 568 goat anti-mouse IgG. The stained embryos were mounted with VECTASHIELD Antifade Mounting Medium (cat#H-1000; VECTOR LABORATORIES). The immunofluorescence images and the AP staining images were taken by a confocal microscope (Zeiss LSM880) and a stereomicroscope (Leica M80), respectively. The image analyses were done by using ImageJ/Fiji software.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!