Tissue-resident intraperitoneal macrophages were harvested as before (22 (link)). After 30 m serum starvation, macrophages were stimulated with media containing 10% serum, then fixed in 4% PFA. Following permeabilization with 0.2% Triton X-100, cells were blocked with 5% normal goat serum in PBS, then stained for the indicated markers. Primary antibodies were anti-CD163 (1:100), anti-CD169 (1:100), and CD209b (1:25), and anti-Talin 1 (1:100); secondary antibodies were Alexa Fluor 594 and Alexa Fluor 647 (1:100). F-actin was illuminated with phalloidin-Alexa Fluor 488 (1:500) and the nucleus with DAPI (1:1000). Images were acquired using Zeiss LSM 880 Airyscan Two-Photon Confocal Microscope.
Protocol detail
Immunofluorescence Analysis of Intraperitoneal Macrophages
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Alexa 594
Alexa fluor 488
Alexa fluor 647
Antibodies
Cd163
Cells
Confocal microscope
Dapi
F actin
Goat
Macrophages
Nucleus
Phalloidin
Serum
Talin
Tissue
Triton x 100
Corresponding Organization :
Other organizations : Washington University in St. Louis
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Variable analysis
independent variables
- Serum starvation duration (30 minutes)
- CD163 expression
- CD169 expression
- CD209b expression
- Talin 1 expression
- F-actin organization
- Nuclear morphology
- Tissue-resident intraperitoneal macrophages
- Cell permeabilization with 0.2% Triton X-100
- Cell blocking with 5% normal goat serum in PBS
- Primary antibodies (anti-CD163, anti-CD169, CD209b, anti-Talin 1)
- Secondary antibodies (Alexa Fluor 594, Alexa Fluor 647)
- F-actin staining with phalloidin-Alexa Fluor 488
- Nuclear staining with DAPI
- Imaging with Zeiss LSM 880 Airyscan Two-Photon Confocal Microscope
- None specified
- None specified
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