Immunofluorescence Staining of CD31



April 8, 2019

EP163 v1.0

Goals:

Key Steps:

Other Required Materials:

Visualize CD31 or platelet endothelial cell adhesion molecule (PECAM-1) in Organ- Chips

  • Immunofluorescence staining in Organ-Chips
  • Anti-CD31 antibody (Abcam ab9498)
  • 4% paraformaldehyde (PFA)
  • 10% saponin
  • PBS
  • BSA
  • Normal goat serum (or other serum from the species the secondary antibody was raised in)
  • Alexa Fluor® 555-conjugated goat anti-mouse IgG secondary antibody (or another anti-mouse secondary antibody)
  • Fluorescence microscope

Background

CD31 or PECAM-1, is a type I integral membrane glycoprotein that is expressed at high levels on endothelial cells. CD31 staining can be used as a marker of endothelial cells. The expression on endothelial cells is concentrated at junctions between adjacent cells. 

Method

Sample type

Fixed Organ-Chip

See Protocol EP173 Fixation and Immunofluorescence (IF) Staining.

Recommended fixative and incubation time

4% PFA, 15 minutes at room temperature

Recommended permeabilization and incubation time

1% saponin in PBS, 30 minutes at room temperature

Recommended blocking buffer and incubation time

1% BSA, 10% goat serum in PBS, overnight at 4°C

Recommended primary antibody dilution and incubation time

Anti-CD31 (PECAM-1) (Abcam ab9498)

1:50 dilution in blocking buffer, overnight at 4°C

Recommended antibody host

Mouse

Recommended secondary antibody dilution and incubation time

Alexa Fluor® 555-conjugated goat anti-mouse IgG secondary antibody 1:500 dilution in blocking buffer, 2 hours at room temperature in the dark.

Representative image

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Image of CD31 (red) / nuclei staining (blue) indicating presence of LSECs in the human Liver-Chip (bottom channel)

More information on vendor site

CD31: https://www.abcam.com/cd31-antibody-jc70a-ab9498.html

 


 

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