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Immunofluorescence Staining Methods


Immunofluorescence staining methods can also be used successfully for labeling multiple anti- gens in the same preparation. These methods are especially useful for co-localization of anti- gens in the same compartment of a cell, and in this regard offer a distinct advantage over enzyme-based detection systems. 

Colon Carcinoma – Double label

Colon (frozen) – Double label
• Multi-Cytokeratin (m), M.O.M.TM Fluorescein Kit (green). TM®
• Desmin (m), M.O.M. Basic Kit, Texas Red Avidin DCS (red).

Traditionally, double fluorescent labeling has been achieved by using fluorescently conjugated secondaries against primary antibodies from different species. Although this technique in general tends to be less sensitive than enzymatic staining, the sensitivity of the fluorescent stain can be increased by using a (strept)avidin-biotin system for amplification of the label. Many of the principles regarding multiple antigen labeling will apply to both immunofluorescent and enzymatic methods (see section on “Immuno- enzymatic Staining Methods”). For best results, sequential staining of each primary antibody is recommended. As in enzymatic applications, the order of labeling, appropriate controls, and additional blocking steps may be important to obtain optimal results. In contrast to enzymatic staining, special consideration must be given to the species of the primary antibodies to be used. For fluorescent applications, the two primary anti- bodies usually should be from different species, otherwise, artifactual co-staining of antigens may result. However, the use of two mouse primary antibodies is the exception. See section on “Multiple Immunofluorescent Labeling Using Two or More Mouse Monoclonal Primary Antibodies”. 

In addition, if the (strept)avidin biotin system is used for the visualization of both antigens, a (strept)avidin-biotin block MUST be used before the second primary antibody step. 

Following labeling it is important to preserve the intensity of the fluorescent signal, as some fluorescent products are prone to rapid fading when viewed under the microscope. In most instances this is easily accomplished by coverslip- ping the preparation with an anti-fade, anti- photobleaching agent such as VECTASHIELD® mounting media. The use of VECTASHIELD® also allows the preparation to be stored for extended periods without significant loss of intensity. 

Autofluorescence of the tissue may obscure staining depending on the type of tissue, the fixation method, and the microscope filter used for visualization. This should be evaluated before staining, and autofluoresence quenched, if necessary, using appropriate methods. 

The following protocols are for frozen tissue sections. These protocols can be adapted for other tissue preparations. 

Tonsil (frozen) – Double label

Tonsil (frozen) – Double label
• Ki67 (m), M.O.M.TM Fluorescein Kit (green).
• Pan-Cytokeratin (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red).

Small Bowel (frozen) – Double label

Small Bowel (frozen) – Double label
• PGP9.5 (m), M.O.M.TM Basic Kit, VECTASTAIN® ABC-AP Standard Kit, Vector® Red AP substrate (red).
• Desmin (m), M.O.M.TM Fluorescein Kit (green).

Protocol: Double Immunofluorescent Labeling Using Two Primary Antibodies From Different Species

Staining for First Antigen

  1. Preparation of tissue. Fix sections with the appropriate fixative for the antigen under study (Please see Note 1).
  2. Air dry sections.
  3. Wash sections 2 x 2 minutes in buffer (PBS).
  4. Avidin/biotin blocking step. Perform Avidin/Biotin blocking if required (Avidin/Biotin Blocking Kit, Cat. No. SP-2001). Incubate sections with Avidin Solution for 15 minutes. Rinse briefly with buffer, then incubate in the Biotin Solution for 15 minutes. Wash sections 2 x 2 minutes in buffer. This blocking step may be eliminated if suitable controls have determined this step to be unnecessary.
  5. Protein blocking step. Incubate sections for 20 minutes with buffer containing 5% normal blocking serum (NS) which was prepared from the species in which the secondary antibody is made.
  6. Blot excess serum from sections.
  7. Primary antibody. Incubate sections with the first primary antibody diluted in appropriate anti- body diluent (buffer containing 5% NS) using an appropriate concentration and length of incubation.
  8. Wash for 5 minutes in buffer.
  9. Secondary antibody. Incubate sections for 30 minutes with biotinylated secondary antibody (5-10 μg/ml diluted in buffer containing 5% NS).
  10. Wash slides for 5 minutes in buffer.
  11. Avidin conjugate. Incubate sections with Fluorescein Avidin DCS (15-20 μg/ml diluted in buffer) for 5-10 minutes.
  12. Wash slides for 5 minutes in buffer. 

    Staining for Second Antigen
  13. Avidin/biotin blocking step. Apply an Avidin/Biotin block according to Step 4. (This step must be done to prevent the interaction of the second set of labeling reagents with the first set of labeling regents).
  14. Protein blocking step. Incubate sections for 20 minutes with 5% NS.
  15. Blot excess serum from sections.
  16. Primary antibody. Incubate sections with second primary antibody diluted in appropriate antibody diluent (buffer containing 5% NS) using an appropriate concentration and length of incubation.
  17. Wash slides for 5 minutes in buffer.
  18. Secondary antibody. Incubate sections for 30 minutes with biotinylated secondary antibody (5-10 μg/ml diluted in buffer containing 5% NS).
  19. Wash slides for 5 minutes in buffer.
  20. Avidin conjugate. Incubate sections with Texas Red® Avidin DCS (15-20 μg/ml diluted in buffer) for 5-10 minutes.
  21. Wash slides for 5 minutes in buffer.
  22. Mount with the appropriate VECTASHIELD® mounting media.
  23. Observe under a fluorescence microscope.

NOTES:
1. Aldehyde-fixed tissues (e.g. formalin) tend to be autofluorescent and may make interpretation of specific fluorescein signal difficult.

Add first primary antibody.
Tonsil (frozen) – Double label

Tonsil (frozen) – Double label
• Ki67 (m), M.O.M.TM Fluorescein Kit (green).
• CD20 (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red).

Multiple Immunofluorescent Labeling using Two or More Mouse Monoclonal Primary Antibodies

Specific localization of a mouse primary antibody on mouse tissue was problematic until the intro- duction of our Vector® Mouse on Mouse (M.O.M.TM) Immunodetection Kits.

These M.O.M.TM kits block endogenous mouse immunoglobulins in mouse tissue, allowing for accurate recognition of the mouse primary antibody by the biotinylated anti-mouse IgG secondary antibody, eliminating confusing background.

Using this same mouse Ig blocking technology, it is possible to fluorescently detect several mouse primary antibodies on the same tissue section whether or not the tissue is of mouse origin. 

No M.O.M Kit Mouse Intestine frozen – Double label

No M.O.MTM. Kit: Mouse Intestine (frozen) – Double label
• Peripherin (m), Biotinylated horse anti-mouse IgG, Fluorescein Avidin DCS (green).
• Desmin (m), Biotinylated horse anti-mouse IgG, Texas Red® Avidin DCS (red).
NOTE BACKGROUND AND SIGNAL MIXING.

With M.O.M.TM Kit: Mouse Intestine (frozen) – Double label

With M.O.M.TM Kit: Mouse Intestine (frozen) – Double label
• Peripherin (m), M.O.M.TM Fluorescein Kit (green).
• Desmin (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red). COMPARE WITH ADJACENT NO M.O.M.TM KIT PHOTO.

No M.O.M.TM Kit: Colon (frozen) – Double label

No M.O.M.TM Kit: Colon (frozen) – Double label
• Multi-Cytokeratin (m), Biotinylated horse anti-mouse IgG, Fluorescein Avidin DCS (green).
• Desmin (m), Biotinylated horse anti-mouse IgG, Texas Red® Avidin DCS (red).
NOTE BACKGROUND AND SIGNAL MIXING.

With M.O.M.TM Kit: Colon (frozen) – Double label

With M.O.M.TM Kit: Colon (frozen) – Double label
• Multi-Cytokeratin (m), M.O.M.TM Fluorescein Kit (green).
• Desmin (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red). COMPARE WITH ADJACENT NO M.O.M.TM KIT PHOTO

 

Protocol: Multiple Immunofluorescent Labeling using Two or More Mouse Monoclonal Primary Antibodies

Staining for First Antigen

    1. Preparation of tissue. Fix sections with the appropriate fixative for the antigen under study (Please see Note 1).
    2. Air dry sections.

    3. Wash sections 2 x 2 minutes in buffer (PBS).
    4. Avidin/biotin blocking step. Perform

Avidin/Biotin blocking if required (Avidin/Biotin Blocking Kit, Cat. No. SP-2001). Incubate sections with Avidin Solution for 15 minutes. Rinse briefly with buffer, then incubate in the Biotin Solution for 15 minutes. Wash sections 2 x 2 minutes in buffer. This blocking step may be eliminated if suitable controls have determined this step to be unnecessary.

  1. Mouse Ig blocking step. Incubate sections for 1 hour in working solution of M.O.M.TM Mouse Ig Blocking Reagent (Please see Note 2).
  2. Wash sections 2 x 2 minutes in buffer (Please see Note 2).
  3. Protein blocking step. Incubate tissue sections for 5 minutes in working solution of M.O.M.TM diluent.
  4. Primary antibody. Tip off excess M.O.M.TM diluent from sections. Dilute primary antibody in M.O.M.TM diluent to the appropriate concen- tration. Incubate section in diluted primary antibody for 30 minutes (Please see Note 3).
  5. . Wash sections 2 x 2 minutes in buffer.
  6. Secondary antibody. Apply working solution of M.O.M.TM Biotinylated Anti-Mouse IgG Reagent. Incubate sections for 10 minutes.
  7. Wash sections 2 x 2 minutes in buffer.
  8. Avidin conjugate. Apply Fluorescein Avidin DCS prepared as described in M.O.M.TM kit instructions. Incubate sections for 5 minutes (Please see Note 4).
  9. Wash sections 2 x 5 minutes in buffer.

    Staining for Second Antigen

  10. Avidin/biotin blocking step. Perform Avidin/Biotin blocking according to step 4. (This step must be done to prevent the interac- tion of the second set of labeling reagents with the first set of labeling reagents).
  11. Mouse Ig blocking step. Incubate sections for 1 hour in working solution of M.O.M.TM Mouse Ig Blocking Reagent.
  12. Wash sections 2 x 2 minutes in buffer.
  13. Protein blocking step. Incubate sections for 5 minutes in working solution of M.O.M.TM diluent.
  14. Primary antibody. Tip off excess M.O.M.TM diluent from sections. Dilute second primary antibody in M.O.M.TM diluent to the appropriate concentration. Incubate section for 30 minutes (Please see Note 3).
  15. Wash sections 2 x 2 minutes in buffer.
  16. Secondary antibody. Apply working solution of M.O.M.TM Biotinylated Anti-Mouse IgG Reagent. Incubate sections for 10 minutes.
  17. Wash sections 2 x 2 minutes in buffer.
  18. Avidin conjugate. Apply Texas Red ® Avidin DCS at a concentration of 15-20 μg/ml in buffer. Incubate sections for 5-10 minutes (Please see Note 4).
  19. Wash sections for 2 x 5 minutes in buffer.
  20. Mount with appropriate VECTASHIELD® mounting media.

NOTES:

1. Aldehyde-fixed tissues (e.g. formalin) tend to be autofluorescent and may make interpretation of specific fluorescein signal difficult.

2. For non-murine tissue, omit step 5 and step 6.

3. Optimal results with the M.O.M.TM kit are usually obtained with a primary antibody incubation of 30 minutes. Primary antibody concentrations should be optimized for multiple labeling applications.

4. Optimal order of the fluorescent label should be determined. Other fluorochrome conjugated streptavidin or avidin reagents can be substituted once optimal signal/noise has been established.

5. A M.O.M.TM Troubleshooting Guide is available online or upon request.

Skeletal Muscle (frozen, unfixed) – Double label

Skeletal Muscle (frozen, unfixed) – Double label
• Alpha-Sarcoglycan (m), M.O.M.TM Fluorescein Kit (green).
• Muscle Specific Actin (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red).

Colon (frozen) – Double label

Colon (frozen) – Double label
• Desmin (m), M.O.M.TM Fluorescein Kit (green).
• Peripherin (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red).

Prostate (frozen) – Double label

Prostate (frozen) – Double label
• Muscle Specific Actin (m), M.O.M.TM Fluorescein Kit (green).
• Multi-Cytokeratin (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red).

Colon (frozen) – Double label

Breast Carcinoma (frozen) – Double label
• Estrogen Receptor (m), M.O.M.TM Fluorescein Kit (green).
• Multi-Cytokeratin (m), M.O.M.TM Basic Kit, VECTASTAIN® ABC-AP Standard Kit, Vector® Red AP substrate (red).

Tonsil (frozen) – Double label

Tonsil (frozen) – Double label
• Ki67 (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red). • CD20 (m), M.O.M.TM Fluorescein Kit (green).
Compare color contrast with adjacent photo.

Tonsil (frozen) – Double label

Tonsil (frozen) – Double label
• Ki67 (m), M.O.M.TM Fluorescein Kit (green).
• CD20 (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS. Compare color contrast with adjacent photo.

Tonsil (frozen) – Double label

Tonsil (frozen) – Double label
• p63 (m), M.O.M.TM Fluorescein Kit (green).
• Multi-Cytokeratin (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red).
Compare color contrast with adjacent photo.

Tonsil (frozen) – Double label

Tonsil (frozen) – Double label
• p63 (m), M.O.M.TM Basic Kit, Texas Red® Avidin DCS (red).
• Multi-Cytokeratin (m), M.O.M.TM Fluorescein Kit (green). Compare color contrast with adjacent photo.