For evaluation of PV biotinylation and localization of EXP1 complementing copies, thin smears were fixed with a mixture of cold 90% acetone and 10% methanol for 2 minutes. For export assays, thin smears were fixed with room temperature 100% acetone for 2 minutes. For EXP2 distribution assays, magnet-purified infected RBCs were allowed to settle on coverslips coated with concanavalin A before fixing with 4% PFA and 0.0075% glutaraldehyde for 15 minutes. IFAs were processed as described (Beck et al., 2014 (link)). For detection of biotinylated proteins, streptavidin-conjugated Alexa Fluor 594 (ThermoFisher) was included with secondary antibodies at 1:200. Images were collected on an Axio Observer 7 equipped with an Axiocam 702 mono camera and Zen 2.6 Pro software (Zeiss) using the same exposure times for all images across sample groups and experimental replicates.
Protocol detail
Immunofluorescence Assays for Malaria
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Acetone
Alexa 594
Antibodies
Assays
Biotinylation
Cold
Concanavalin a
Exp1
Glutaraldehyde
Ifas
Methanol
Proteins
Rbcs
Streptavidin
Corresponding Organization : Washington University in St. Louis
Other organizations : University of California, Los Angeles
Top 5 similar protocols
Variable analysis
independent variables
- Fixation of thin smears: 90% acetone and 10% methanol for 2 minutes
- Fixation of thin smears: 100% acetone at room temperature for 2 minutes
- Fixation of magnet-purified infected RBCs: 4% PFA and 0.0075% glutaraldehyde for 15 minutes
- Localization of EXP1 complementing copies
- Export assays
- EXP2 distribution assays
- Coated coverslips with concanavalin A for magnet-purified infected RBCs
- Exposure time for image collection was kept the same across sample groups and experimental replicates
- Positive control: Not specified
- Negative control: Not specified
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!