Human SSC line was fixed with 4% paraformaldehyde (PFA, room temperature, 30 min), permeabilized with 0.5% Triton X-100 (Sigma), blocked with 5% BSA (room temperature, 1 h) and incubated with the primary antibodies against FOXP3 (Santa Cruz, sc-166212) at a dilution of 1:50 at 4°C overnight. Antibody binding was detected with Alexa Fluor-conjugated second antibody (Invitrogen, USA) (1:200, room temperature, 1 h) and the nuclei were stained with DAPI (room temperature, 5 min). After extensive washes with PBS, cells were observed under the fluorescence microscope.
Alexa fluor conjugated second antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor-conjugated second antibody is a secondary antibody that is labeled with an Alexa Fluor dye. It is designed to be used in immunoassays and other applications that require the detection of primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Axio observer, by Zeiss (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti uch l1, by Abcam (1 mentions)
Anti foxp3, by Abcam (1 mentions)
Anti pcna, by Abcam (1 mentions)
Nis elements ar 3, by Nikon (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
5 protocols using alexa fluor conjugated second antibody
1
FOXP3 Protein Immunofluorescence Staining
2
Immunohistochemical Analysis of Human Testis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human testis paraffin sections were routinely deparaffinized and hydrated. After antigen retrieval and endogenous peroxidase blocking, the sections were blocked with 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at room temperature and incubated with primary antibodies, including anti-FOXP3 (Abcam, catalogue: ab22510), anti-UCHL1 (Abcam), anti-PCNA (Abcam), and anti-Ki67 (Abcam), at 4°C overnight. Antibodies binding were detected with HRP conjugated second antibodies, stained by DAB (Vector Lab, Burlingame, USA) and counterstained with hematoxylin, or detected by Alexa Fluor-conjugated second antibody (Invitrogen, USA) (1:200, room temperature, 1 h). DAPI was utilized for staining cell nuclei. The images were captured using the fluorescence microscope (Nikon, Tokyo, Japan).
3
Visualizing TGFβ Receptor Trafficking
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HSCs or murine tissue sections were fixed with 4% paraformaldehyde followed by permeabilization by 0.2% Triton X-100 and a primary antibody was applied. After incubation with a primary antibody for 2 hours at room temperature or overnight at 4°C, an Alexa Fluor-conjugated second antibody (Thermo Fisher Scientific, Waltham, MA) was used for IF labeling and detection. Cell nuclei were counterstained with DAPI (D1306; Thermo Fisher Scientific). IF signals were captured by an Axio observer (Zeiss, Oberkochen, Germany) with or without ApoTome function.32 (link)To analyze the sorting of TβRII to early endosomes or lysosomes induced by TGFβ1, HSCs expressing TβRII-HA were serum-starved and pretreated with 40 αg/mL of cycloheximide for 1 hour to block the synthesis of proteins including TβRII-HA. TGFβ1 was added in and TGFβ1/receptor binding was permitted by incubating the cells for 30 minutes at 4°C. After washing off free TGFβ1, cells were incubated at 37°C for various times and harvested for double IF for EEA-1/TβRII-HA or LAMP1/TβRII-HA. IF was captured by an Axio observer using a 63x lens with ApoTome function. The rate of TβRII-HA/EEA-1 and TβRII-HA/LAMP1 colocalization was analyzed as we previously described.1 (link)
4
Visualizing TGFβ Receptor Trafficking
5
BrU Labeling of Untreated and siRNA Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The BrU labeling protocol was as described previously (Wang et al., 2008 (link)). Untreated or siRNA-transfected cells were labeled with five-bromouridine (BrU) at 0.2 mM for 5 min. The incorporation was measured by staining with an anti-BrdU antibody that also recognizes BrU (Sigma, catalogue# B8434) at a 1:50 dilution, followed by an Alexa Fluor-conjugated second antibody (Thermo Fisher). Images were obtained with a Nikon Eclipse Ti-E inverted fluorescence microscope using NIS-Elements AR 3.2 software (Nikon).
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!