Immunofluorescence Staining of Alpha Smooth Muscle Actin



April 4, 2019

EP136 v1.0

Goals:

Key Steps:

Other Required Materials:

Visualize smooth-muscle actin filaments in fixed Emulate Organ-Chip

  • Immunofluorescence staining in chip
  • Anti-alpha smooth muscle actin (α-SMA) antibody (Abcam, ab5694)
  • 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 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-α-SMA antibody (Abcam, ab5694)

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 α-SMA / nuclei staining indicating activated stellate cells in the human Liver-Chip (bottom channel)

More information on vendor site

https://www.abcam.com/alpha-smooth-muscle-actin-antibody-ab5694.html?productWallTab=ShowAll

 


 

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The technology disclosed in this document may be covered by one or more patents or patent applications owned by or licensed to Emulate, Inc. No license is granted herein. Further information is available by contacting Emulate.