For immunofluorescence, 1 × 105 cells were seeded in 24 well-plates containing gelatin-coated coverslips and they were fixed after 24 h using 4% paraformaldehyde in PBS 1X for 15 min at 4 °C. Cells were permeabilized using PBS1x-20%FBS containing 0.2% Triton X-100 for 30 min at room temperature. Then, blocking was performed using 5% BSA in PBS 1X for 1 h. The primary antibodies were goat IgG GRP94 (1/800, sc-1794, Santa Cruz Biotechnology), mouse IgM GM2 (1/50, A2582, Tokyo Chemical Industry), mouse IgM GM3 (1/150, A2576, Tokyo Chemical Industry) and rabbit IgG LC3B (1/500, 3868, Cell Signaling). Alexa Fluor® 555 anti-goat IgG, Dylight® 650 anti-mouse IgM and Alexa Fluor® 594 anti-rabbit IgG (Invitrogen) were used as secondary antibodies (1/1000). Hoescht 33342 staining (Invitrogen) was used for nucleus visualization. For colocalization experiments with Lysotracker (Invitrogen), cells were pre-incubated for 90 minutes with a 75 nM solution of Lysotracker in culture medium before starting the immunofluorescence protocol.
Sc 1794
Manufactured by Santa Cruz Biotechnology
Sc-1794 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for general laboratory use, but its core function is not specified in detail. A more detailed and unbiased description is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Seeblue plus2 pre stained standard, by Thermo Fisher Scientific (1 mentions)
Gapdh, by BD (1 mentions)
Anti protease cocktail, by Roche (1 mentions)
Grp94, by Santa Cruz Biotechnology (1 mentions)
Af8047, by R&D Systems (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
3 protocols using sc 1794
1
Immunofluorescence Staining of Cellular Organelles
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in a 1% SDS (v/v) extraction buffer containing an anti-protease cocktail (Roche). Protein concentrations were determined using the Bradford assay (MicroBCA, Pierce). After resolution by SDS-PAGE, electrophoresed proteins were transferred to polyvinylidene fluoride (PVDF) membranes that were blocked and probed with the following antibodies: GRP94 (1:200, sc1794, Santa Cruz Biotechnology), HexA (1:100, sc-376777, Santa Cruz Biotechnology), GM2-AP (1:100, sc-514437, Santa Cruz Biotechnology), GAPDH (1:2500, BD Pharmingen) and the corresponding peroxidase-conjugated secondary antibodies at 1:2000. Immunoreactive bands were quantified using a VersaDoc™ (Bio-Rad) Imaging System using the Super Signal west-Pico (Pierce). Molecular weights were established with See Blue Plus2 Pre-stained Standard (Invitrogen).
3
Intracellular Localization of CRELD2 in Chondrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess the intracellular localization of CRELD2, primary chondrocytes were fixed in 10% formalin for 10 minutes at room temperature. Plasma membranes were labeled with wheat germ agglutinin (WGA) AlexaFluor 594 conjugate. Cells were then permeabilized for 5 minutes in 0.5% Triton-X before immunolabeling. The following primary antibodies were used: CRELD2, GRP94 (sc1794, Santa Cruz), GLG1 (AF7879, Biotechne), and EEA1 (AF8047, R&D Systems). For labeling of membrane LRP1, living cells were incubated for 2 hours at 37 C in a primary antibody raised against LRP-1 clone 8G1 that detects the extracellular LRP1 α-chain (Abcam). The cells were washed three times in PBS and incubated in an anti-mouse secondary antibody labeled with AlexaFluor 488 for 20 minutes at 13 C. Cells were then fixed in 10% formalin for 10 minutes at room temperature. Immunocytochemistry slides were mounted in Fluoroshield mounting media with DAPI and images were acquired at ×630 magnification using a Nikon (Tokyo, Japan) A1R confocal microscope.
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!