Immunofluorescence Staining of Vimentin



April 23, 2019

EP150 v1.0

 

Goals:

Key Steps:

Other Required Materials:

Visualize vimentin via fluorescent imaging in fixed Emulate Organ-Chips

  • Immunofluorescence staining in Organ-Chips
  • Anti-vimentin antibody [SP20] (Abcam ab16700) 4% paraformaldehyde (PFA)
  • 10% Saponin
  • PBS
  • BSA
  • Normal goat serum (or other serum from the species the secondary antibody was raised in)
  • Alexa Fluor® 488-conjugated goat anti-rabbit IgG secondary antibody (or other anti-rabbit secondary)
  • Fluorescence microscope

Method

Sample type

Fixed Organ-Chip

See Emulate Protocol EP137 Fixation and Immunofluorescence (IF) Staining.

Recommended fixative and incubation time

4% PFA, 15 mins 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-Vimentin [SP20] (Abcam ab16700)

1:1000 dilution in blocking buffer, overnight at 4°C

Recommended antibody host

Rabbit

Recommended secondary antibody dilution and incubation time

Alexa Fluor® 488-conjugated goat anti-rabbit IgG secondary antibody 1:500 dilution in blocking buffer, 2 hours at room temperature in the dark.

Representative image

Image of human stellate cells stained by vimentin (green) in the human Liver-Chip (bottom channel)

More information on vendor site

https://www.abcam.com/vimentin-antibody-sp20-ab16700.html

 


 

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