Mice were infected with H37Rv Mtb-mCherry and sacrificed at D10 and D14. Lungs were excised and submerged in BD Cytofix fixative solution diluted 1:3 with PBS for 24hr at 4°C. Lungs were washed 2x in PBS and dehydrated in 30% sucrose for 24hr prior to OCT embedding and rapid freezing in a methylbutane-dry ice slurry. 20um sections were stained overnight at RT (with the following antibodies: CD11c BV480; HL3 (BD), CD11b BV510; M1/70 (BD), CD45.2 AF700; 104 (BioLegend), MHC-II APC-Fire750; M5/114.15.2 (BioLegend), Siglec-F BV421; E50-2440 (BD), pS6 AF488; 2F9 (Cell Signaling), CD3 CF633; 17A2, CD4 CF660C; RM4-5) and coverslipped with Fluoromount G mounting media (Southern Biotec). Images were acquired on a Leica SP8X confocal microscope, compensated for fluorophore spillover using LAS X (Leica), and analyzed with Imaris (Bitplane) and FlowJo (11 (link)). More details can be found in Supplemental Fig. 3 .
Protocol detail
Mycobacterium tuberculosis Infection in Mice
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.
Corresponding Organization : University of Washington
Top 5 similar protocols
Variable analysis
independent variables
- Days post-infection (D10 and D14)
- Lung cellular composition (CD11c, CD11b, CD45.2, MHC-II, Siglec-F, pS6, CD3, CD4)
- Mtb strain (H37Rv Mtb-mCherry)
- Fixation and dehydration procedure (BD Cytofix fixative solution, 30% sucrose)
- Staining antibodies (CD11c BV480, CD11b BV510, CD45.2 AF700, MHC-II APC-Fire750, Siglec-F BV421, pS6 AF488, CD3 CF633, CD4 CF660C)
- Imaging method (Leica SP8X confocal microscope)
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!