/  Immunohistochemistry   How do I label multiple antigens in the same tissue section?
      

A number of enzymatic methods are available for the immunohistochemical localization of two or more antigens in the same tissue section. These methods fall into two main categories:

Use of the same enzyme system with different substrates to detect each antigen

Use of different enzyme systems and their substrates to label each antigen

The wide range of VECTASTAIN® ABC kits and substrate kits currently offered can be used for either method. Several of the unique substrates developed by Vector are particularly useful for multiple labeling.

In general, the protocol illustrated here is useful for formalin-fixed, paraffin-embedded or frozen tissue sections and primary antibodies raised in one or more species. Most of the photographs show mouse monoclonal antibodies used for both the first and second label.

The preferred method for double or triple labeling involves sequential staining of each primary antibody. The order of labeling, several controls, and additional blocking steps, may be important to obtain optimal results.

Basic Protocol for Double or Triple Label Immunostaining using the VECTASTAIN ABC systems

1. Deparaffinize and rehydrate tissue sections. Rinse in distilled water for 5 minutes.

2. If endogenous peroxidase and/or alkaline phosphatase activities are present in the section, appropriate inactivation methods should be employed.

3. Wash sections in 10 mM sodium phosphate buffer, pH 7.5, 150 mM NaCl (PBS) for 2x3 min. (Other buffers like Tris buffered saline may also be used).

(All incubations are at room teperature unless otherwise noted.)

Staining for First Antigen

4. Blocking Step. Incubate sections for 20 min with PBS containing 5% normal serum (NS) prepared from the 1st VECTASTAIN® ABC Kit or incubate for 5-10 min in 10% NS.

5. Primary Antibody. Blot excess blocking solution from sections and incubate with the 1st primary antibody diluted in 5% NS from the 1st VECTASTAIN® ABC Kit using appropriate concentration and length of incubation. Higher concentrations of primary antibody allow for shorter incubation times. Wash sections in PBS for 2 x 3 min.

6. Secondary Antibody. Incubate sections for 30 min with biotinylated secondary antibody from the 1st VECTASTAIN® ABC Kit diluted in 5% NS. For a 10 min incubation, double the concentration of the biotinylated antibody and normal serum. Wash sections in PBS for 2 x 3 min.

7. ABC. Incubate sections for 30 min with the 1st VECTASTAIN® ABC Reagent prepared in advance as described in the kit instructions. For a 5-10 min incubation, use the VECTASTAIN® ABC Reagent at twice the recommended concentration. Wash sections in PBS for 2 x 3 min.

8. Substrate. Incubate sections with appropriate enzyme substrate until optimal color develops. Use the recommended times given in the substrate kit instructions as a guideline. Wash sections in PBS for 2 x 3 min. Proceed with protocol to stain the second antigen.

If the staining protocol must be interrupted, sections may be kept in PBS at 4 °C until staining is resumed.

Staining for Second Antigen

9. Blocking Step. Incubate sections for 20 min with PBS containing 5% normal serum (NS) from the 2nd VECTASTAIN® ABC Kit or incubate for 5-10 min in 10% NS.

10. Primary Antibody*. Blot excess blocking solution from sections and incubate with the 2nd primary antibody diluted in 5% NS from the 2nd VECTASTAIN® ABC Kit using appropriate concentration and length of incubation. Higher concentrations of primary antibody allow for shorter incubation times. Wash sections in PBS for 2 x 3 min.

11. Secondary Antibody*. Incubate sections for 30 min with biotinylated secondary antibody appropriate for labeling the 2nd primary antibody diluted in 5% NS. For a 10 min incubation, double the concentration of the biotinylated antibody and normal serum. Wash sections in PBS for 2 x 3 min.

12. ABC**. Incubate sections for 30 min with the 2nd VECTASTAIN® ABC Reagent prepared in advance as described in the kit instructions. For a 5-10 min incubation, use the VECTASTAIN® ABC Reagent at twice the recommended concentration. Wash sections in PBS for 2 x 3 min.

13. Substrate. Incubate sections with appropriate enzyme substrate until optimal color develops. Use the recommended times given in the substrate kit instructions as a guideline. Wash sections in tap water for 5 min. Counterstain (if desired) and coverslip using the appropriate protocol for aqueous or permanent mounting.

*If both primary antibodies have been made in the same species (eg. two mouse monoclonal antibodies), the normal serum and biotinylated antibody from the first VECTASTAIN® ABC Kit can be used for the second label.

**The 1st VECTASTAIN® ABC Reagent can also be used for staining the second antigen if the same enzyme is desired.

A third antigen can be detected (triple labeling) by simply continuing the same protocol with an additional staining sequence appropriate for the third primary antibody.

This protocol provides only the basic techniques and further manipulation may be required to optimize staining in individual cases.

Interrupting the Staining Protocol

Multiple labeling can increase the time required for staining antigens. If necessary, choose an appropriate stopping point during the staining protocol. Generally we recommend that if the staining protocol must be interrupted, it should be after substrate development of the first antigen. Sections usually can be kept in PBS at 4 °C until staining is resumed. During any pause in the protocol, sections should be kept moist and in a condition that will preserve antigens.