Immunofluorescence

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Introduction

The Image-iT® Fix Perm Kit contains all the components needed to prepare cells for labeling with antibodies, and fluorescent dyes. The room temperature stable kit contains fixative, permeabilization solution, blocking solution and wash buffer. All reagents come in ready to use 1 x concentrations that are conveniently stored together at room temperature. This protocol provides the steps necessary for using the Image-iT® Fixation/Permeabilization Kit (R37602) for immunofluorescence protocols using primary antibodies and fluorescent dyes.

Materials

  • Image-iT® Fixation/Permeabilization Kit (Cat. #R37602) containing:
  • Wash Buffer
  • Fixative Solution
  • Permeabilization Solution
  • Blocking Solution
  • Anti-PMP70 primary antibody (Cat. #71-8300)
  • Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (Cat. #A11034)
  • Alexa Fluor® 594-phalloidin (Cat. #A12381)
  • NucBlue™ Fixed Cell Stain (Cat. #R37606)
  • ProLong® Gold Antifade Reagent (Cat. #P36930)
  • 6-well plate or 35mm dish (protocol designed for ~10 cm2 growth area)

Protocol

1)     Fixation of cells: Remove media
2)     Add 1 mL Fixative Solution. 
3)     Incubate at room temperature for 15 minutes.
4)     Wash 3x with 2 mL of Wash Buffer for 2-5 minutes.
5)     Permeabilization of cells: Remove wash buffer
6)     Add 1 mL Permeabilization Solution. 
7)     Incubate at room temperature for 15 minutes.
8)     Wash 3x with 2 mL of Wash Buffer for 2-5 minutes.
9)     Blocking of Cells: Remove wash buffer
10)    Add 1 mL of Blocking Solution
11)    Incubate at room temperature for 1 hour
12)    Primary antibody labeling of cells: Remove blocking solution
13)    To stain peroxisomes, add 1 mL anti-PMP70 primary antibody diluted 1:1000 in Blocking Solution
14)    Incubate at room temperature for 1 hour
15)    Wash 3x with 2 mL of Wash Buffer for 2-5 minutes.
16)    Secondary antibody labeling of cells: Remove wash solution
17)    Add 1 mL Alexa Fluor® 488 goat anti-rabbit secondary antibody diluted 1:1000 in Wash Buffer.
18)    Incubate at room temperature for 1 hour in dark.
19)    Wash 3x with 2 mL of Wash Buffer for 2-5 minutes.
20)    Actin staining: Add 1 mL of Alexa Fluor® 594 Phalloidin diluted 1:100 in Wash Buffer. 
21)    Incubate at room temperature for 20 minutes.
22)    Wash 3x with 2 mL of Wash Buffer for 2-5 minutes
23)    Remove wash buffer
24)    Nuclear staining: Add 2 mL wash buffer
25)    Add 4 drops of NucBlue™ Fixed Cell Stain to directly to wash buffer
26)    Incubate at room temperature for 10 minutes.
27)    Wash 3x with 2 mL of Wash Buffer for 2-5 minutes.
28)    Mounting: Remove wash buffer
29)    Mount with ProLong® Gold mounting media
30)    Allow to cure overnight at room temperature
31)    Image using DAPI (blue), FITC (green), and Texas Red® (red) filters

 

HDFn cells labeled with anti-PMP70 (peroxisomes

HDFn cells labeled with anti-PMP70 (peroxisomes) with Alexa Fluor® 488 secondary antibody (green) following fixation and permeabilization using the Image-iT® Fixation/Permeabilization Kit (R37602).

 

LT176   2012-06-08