Immunofluorescence Staining of CD68



April 9, 2019

EP156 v1.0

Goals:

Key Steps:

Other Required Materials:

Visualize CD68, a Kupffer cell marker, in fixed Emulate Organ-Chip

  • Immunofluorescence staining in chip
  • Anti-CD68 monoclonal antibody (KP1) (InvitrogenTM, MA5-13324)
  • 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 other anti-mouse secondary antibody)
  • Fluorescence microscope

Method

Sample type

Fixed Organ-Chip

See Protocol EP137 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-CD68 monoclonal antibody (KP1) (InvitrogenTM MA5-13324)

1:100 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

../../../../../../Volumes/GAURI'S%20USB/5-31-18%20KC,HEP%20LOT%20TEST/K Image of CD68 (red) / nuclei staining (blue) indicating presence of Kupffer cells in the human Liver-Chip (bottom channel)

More information on vendor site

https://www.thermofisher.com/antibody/product/CD68-Antibody-clone-KP1-Monoclonal/MA5-13324


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InvitrogenTM and Alexa Fluor™ are trademarks of Thermo Fisher Scientific or its subsidiaries.

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