Immunofluorescence Microscopy of Parasites

Confluent HFF cultures on glass coverslips were infected with parasites for the indicated times and under specified growth conditions. Monolayers were fixed, permeabilized, and incubated with antibody as previously described (51 (link)). The following primary antibodies were used: rat monoclonal α-HA (clone 3F10; Roche Applied Sciences), rabbit α-Myc (clone 71D10; Cell Signaling Technology), mouse α-Centrin (clone 20H5; Millipore Sigma), mouse α-CenH3 (centromere marker; kindly provided by Boris Striepen, University of Pennsylvania, Philadelphia, PA), mouse α-Ndc80 (outer kinetochore marker; kindly provided by Marc-Jan Gubbels, Boston College, MA), rabbit α-MORN1 (centrocone and basal complex stains; kindly provided by Marc-Jan Gubbels, Boston College, MA), and rabbit and mouse α-IMC1 (parasite shape and internal daughter bud strains; kindly provided by Gary Ward, University of Vermont, VT). All Alexa-conjugated secondary antibodies (Molecular Probes, Life Technologies) were used at a dilution of 1:500. Coverslips were mounted with Aquamount (Thermo Scientific), dried overnight at 4°C, and viewed on a Zeiss Axiovert microscope equipped with a 100× objective using the ApoTome slicer. The collected images were processed first using Zeiss Zen software and then in Adobe Photoshop 2020 using linear adjustment when needed.

Free full text: Click here

Hawkins L.M., Naumov A.V., Batra M., Wang C., Chaput D, & Suvorova E.S. (2022). Novel CRK-Cyclin Complex Controls Spindle Assembly Checkpoint in Toxoplasma Endodyogeny. mBio, 13(1), e03561-21.

Publication 2022

rat monoclonal α-HA (clone 3F10; Alexa-conjugated secondary antibodies Aquamount Axiovert microscope

Antibodies Antibody Centrin Centromere Clone Daughter Dilution Growth conditions Kinetochore Marc Microscope Molecular probes Mouse Ndc80 Parasite Rabbit Stains Strains

Corresponding Organization : University of South Florida

Top 5 similar protocols

Protocol cited in 1 other protocol

Variable analysis

independent variables

Infection time
Growth conditions

dependent variables

Parasite localization and distribution (as measured by immunofluorescence staining)

control variables

HFF cell culture conditions
Antibody staining and imaging protocols

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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.

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?

Revolutionizing how scientists
search and build protocols!