Prestige Antibody Immunofluorescence Procedure

Product Description

A large number of the Prestige Antibodies^® have been used in subcellular localization studies by immunofluorescence (IF) staining of three cell lines: A-431, U-2 OS, and U-251MG. Each cell line is stained with the Prestige Antibody, two organelle probes specific for the endoplasmic reticulum and microtubules, as well as counterstained with the nuclear probe DAPI.

Preparation Instructions

1´ PBS (P5368) – 8.1 mM Na₂HPO₄ (S3264), 1.5 mM KH₂PO₄ (P9791), 137 mM NaCl (S3014), and 2.7 mM KCl (P9541), pH 7.2

Sample Preparation

All washes are performed at room temperature.

1. A multiwell, glass-bottomed, plate is coated with fibronectin (concentration 12.5 µg/ml) for 1 hour at room temperature.
   
2. Cells are seeded (10 000-15 000 cells per well) and incubated at 37°C in humified air with 5.2% CO₂, for at least 4 hours.

Immunostaining

3. Growth medium is removed and the cells are washed in PBS (8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.2).
4. The cells are fixed for 15 minutes in ice cold 4% paraformaldehyde pH 7.2-7.3 in growth medium supplemented with 10% fetal bovine serum (FBS).
5. The cells are permeabilized 3x5 minutes with 0.1% Triton X-100 in PBS.
6. The cells are washed with PBS and incubated overnight at 4°C with primary antibodies in PBS supplemented with 4% FBS.
7. The following day the cells are washed 4x10 minutes with PBS and incubated for 1.5 hours in room temperature with secondary antibodies in PBS supplemented with 4% FBS.
   
   NOTE: The secondary antibodies are fluorescently labeled and thus light sensitive. The sample should be kept in dim light in this as well as in the following steps.
   
   
8. The cells are counterstained for 4 minutes with the nuclear stain DAPI 0.6 µM in PBS.
9. The cells are washed 4x10 minutes with PBS and mounted in glycerol +10% 10xPBS.

Reagents

Primary Antibodies:

• Prestige Antibodies at a working concentration of 1-4 µg/ml.
  
  NOTE: The dilution of the primary antibody is to be considered as a guideline only. Optimal dilution must be determined by the user.
  
  
• Chicken anti-Calreticulin polyclonal antibody
• Mouse anti-alpha Tubulin monoclonal antibody

Secondary Aantibodies:

• Alexa^® Fluor 555 goat anti-mouse IgG
• Alexa^® Fluor 647 goat anti-chicken IgG
• Alexa^® Fluor 488 goat anti-rabbit IgG

Prestige Antibodies is a registered trademark of Sigma- Aldrich^® Biotechnology LP and Sigma-Aldrich Co. LLC
Alexa Fluor is a registered trademark of Molecular Probes.
TRITON is a registered trademark of Union Carbide Corp.

View our full line of Prestige Antibodies^®