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Multiple Antigen Labeling


The preferred method for double or triple labeling involves sequential staining of each primary antibody. With proper color development of substrate, the reaction product from the first substrate usually prevents most of the subsequent antibodies and detection reagent(s) from interacting with components used to stain the first antigen. This feature allows multiple labeling with primary antibodies raised in the same species using a single VECTASTAIN® ABC kit or ImmPRESS™ reagent. The order of the primary antibodies and the substrates is very important. Several controls and additional blocking steps may be necessary to obtain optimal results. Numerous combinations of colors are possible using Vector Laboratories’ products and our multiple labeling protocols.

Detection by electron microscopy of a plasmid biotinylated

Colon – Double label
• M2A Antigen (m), ImmPRESS™ Universal Reagent, DAB+Ni HRP substrate (gray/black).
• Cytokeratin 8/18 (m), ImmPRESS™ Universal Reagent, Vector® NovaRED™ HRP substrate (red).

The following protocols are for formalin-fixed, paraffin-embedded tissue. These protocols can be adapted for other tissue preparations.

Order of Labeling for Primary Antibodies

  • The order of labeling may significantly affect the quality and labeling pattern of each antigen in the stained section.
  • Once the detection systems and substrates have been selected, it is best to try single labeling protocols for each of the primary antibodies with each labeling system.
  • Optimize the staining conditions for each primary antibody. Include any necessary pretreatments such as high temperature antigen unmasking or proteolytic digestion and titer the antibodies to find the optimal dilutions and incubation times. All pretreatments must be appropriate for the staining of all subsequent antigens in the procedure. Once optimized, the same conditions should then be used in the double labeling protocol.
  • Use sections stained with the optimized single label conditions as controls to compare the quality of staining and the labeling pattern for each antigen in the double label protocol.
  • To verify that steric hindrance is not adversely affecting the labeling of the second antigen, first perform the double label protocol using one sequence of the two primary antibodies. Then, keeping the order of the detection systems the same, reverse the sequence of the two primary antibodies. Select the protocol giving the best labeling patterns. For example, nuclear antigens are generally best labeled before cytoplasmic antigens, and cell membrane antigens are often best labeled before cytoplasmic antigens
Tumor – Double Label

Tumor – Double Label
• p53 protein (m), VECTASTAIN® ABC-AP Kit, Vector® Red AP substrate (red).
• Pan-Cytokeratin (s), VECTASTAIN® Elite® ABC Kit, Vector® SG HRP substrate (blue/gray).

Endometrium – Double label

Endometrium – Double label
• Progesterone Receptor (rm), VECTASTAIN® Universal ABCAP Kit, Vector® Blue AP substrate (blue).
• CD34 (m), VECTASTAIN® Universal Elite® ABC Kit, DAB HRP substrate (brown).

Order of Substrates in Labeling Protocol This Enzyme Substrate Combinations Table is designed as a reference for optimal multiple labeling, because the order of the two colored precipitates can significantly affect the quality, color, and labeling pattern of each antigen in the stained section. This chart ensures that distinct colors are visible after the labeling reactions are completed using an optimized multiple labeling protocol.

Enzyme Substrate Combinations

Second Substrate
First Substrate
Vector® Red
(red)
Vector® Blue
(blue)
Vector® Black
(brown/black)
BCIP/NBT 
(blue/violet)
Vector® VIP 
(purple)
DAB 
(brown)
DAB-Ni 
(gray/black)
Vector® 
NovaRED™ (red)
Vector® SG 
(blue/gray)
AEC
(red)
TMB*
(blue)
Vector® Red (red) 
Cat. No. SK-5100
   -  -  -  -  +  +  -  +  -  -
Vector® Blue (blue) 
Cat. No. SK-5300
 +    -  -  +  +  +  +  +  +  -
Vector® Black (brown/black) 
Cat. No. Sk-5200
 +  +    +  +  -  -  -  -  +  -
 BCIP/NBT (blue/violet) 
Cat. No. SK-5400
 +  -  -    +  +  +  +  +  +  -
Vector® VIP (purple) 
Cat. No. SK-4600
 -  +  -  -    +  +  -  +  -  -
DAB (brown) 
Cat. No. SK-4100
 +  +  -  +  +    -  -  +  +  +
DAB-Ni (gray/black) 
Cat. No. SK-4100
 +  -  -  -  +  +    +  -  +  +
Vector® NovaRED™ (red) 
Cat. No. SK-4800
 -  +  -  +  -  +  +    +  -  +
Vector® SG (blue/gray) 
Cat. No. Sk-4700
 +  -  -  -  +  +  -  -    +  -
AEC (red) 
Cat. No. SK-4200
 -  -  -  -  -  +  -  -  +    -
TMB* (blue) 
Cat. No. SK-4400
 -  -  -  -  -  -  -  -  -  -  
KEY:
Alkaline Phosphatase Peroxidase
+ indicates good contrast,
– indicates incompatibility of substrates for various reasons
* TMB does not provide optimal reactivity in double label applications.

 

Protocol: Multiple Antigen Labeling Using the VECTASTAIN® Systems

Staining for First Antigen

Add First Primary Antibody
Add First Primary Antibody
  1. Preparation of tissue. Deparaffinize and rehydrate tissue sections following standard protocols.
  2. Rinse in distilled water for 5 minutes.
  3. If endogenous enzyme activities are present inactivate using appropriate methods (See Appendix 2).
  4. Wash sections 2 x 3 minutes in 10 mM sodium phosphate buffer, pH 7.5, 150 mM NaCI (PBS). (Other buffers may be used).
  5. 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 such background not to be a concern.
  6. Protein blocking step. Incubate sections for 20 minutes with buffer containing 5% normal serum (NS) prepared from the first VECTASTAIN® kit, or incubate for 5-10 minutes in 10% NS.
  7. Primary antibody. Blot excess blocking solution from sections and incubate with the first primary antibody diluted in 5% NS from the first VECTASTAIN® kit using appropriate concentration and length of incubation.
  8. Wash 2 x 3 minutes in buffer.
  9. Secondary antibody. Incubate sections for 30 minutes with biotinylated secondary antibody from the first VECTASTAIN® kit diluted in 5% NS. For a 5-10 minute incubation, double the concentration of the biotinylated antibody and normal serum.
  10. Wash sections 2 x 3 minutes in buffer.
  11. ABC. Incubate sections for 30 minutes with the first VECTASTAIN® ABC reagent prepared in advance as described in the kit instructions. For a 5-10 minute incubation, use the VECTASTAIN® ABC reagent at twice the recommended concentration.
  12. Wash sections 2 x 3 minutes in buffer.
  13. Substrate. Incubate sections with the appropriate enzyme substrate until optimal color develops. Use the recommended times given in the substrate kit instructions as a guideline.
  14. Wash sections 2 x 3 minutes in buffer.

    Staining for Second Antigen

  15. Avidin/biotin blocking step. Perform Avidin/Biotin blocking step if required. (This step may be necessary to prevent the interaction of the second set of labeling reagents with the first set of labeling reagents.)
  16. Protein blocking step. Incubate sections for 20 minutes with buffer containing 5% NS prepared from the second VECTASTAIN® kit, or incubate for 5-10 minutes in 10% NS.
  17. Primary antibody. Blot excess blocking solution from sections and incubate with the second primary antibody diluted in 5% NS from the second VECTASTAIN® kit using appropriate concentration and length of incubation.
  18. Wash 2 x 3 minutes in buffer.
  19. Secondary antibody. Incubate sections for 30 minutes with biotinylated secondary antibody appropriate for labeling the second primary antibody diluted in 5% NS. For a 5-10 minute incubation, double the concentration of the biotinylated antibody and normal serum.
  20. ABC. Incubate sections for 30 minutes with the second VECTASTAIN® ABC reagent prepared in advance as described in the kit instructions. For a 5-10 minute incubation, use the second VECTASTAIN® ABC reagent at twice the recommended concentration.
  21. Wash sections 2 x 3 minutes in buffer
  22. Substrate. Incubate sections with the appropriate second, contrasting enzyme substrate until optimal color develops. Use the recommended times given in the substrate kit instructions as a guideline.
  23. Wash sections in tap water for 5 minutes.
  24. Counterstain, clear, and mount in appropriate mounting medium.

Protocol: Multiple Antigen Labeling Using the ImmPRESS™ Reagents

Staining for First Antigen

  1. Preparation of tissue. Deparaffinize and rehydrate tissue sections following standard protocols.
  2. Rinse in distilled water for 5 minutes.
  3. If endogenous peroxidase activity is present in the section, inactivate using an appropriate method (See Appendix 2).
  4. Wash sections 2 x 3 minutes in 10 mM sodium phosphate buffer, pH 7.5, 150 mM NaCI (PBS). (Other buffers may be used).
  5. Protein blocking step. Incubate sections for 20 minutes with ready-to-use (2.5%) normal horse serum (NHS).
  6. Primary antibody. Incubate sections with mouse or rabbit primary antibody diluted in appropriate antibody diluent (buffer containing diluted (2.5%) NHS or 0.1% immunohistochemical grade bovine serum albumin (BSA)).
  7. Wash slides for 5 minutes in buffer.
  8. ImmPRESS™ Reagent. Incubate sections for 30 minutes with appropriate ImmPRESS™ Reagent (anti-mouse Ig, anti-rabbit Ig, or universal).
  9. Wash slides for 5 minutes in buffer.
  10. 1Substrate. Incubate sections in peroxidase substrate solution until optimal color develops.
  11.  Rinse for 5 minutes in buffer.

    Staining for Second Antigen

  12.  Protein blocking step. Incubate sections for 20 minutes with ready-to-use NHS.
  13. Primary antibody. Incubate sections with the second mouse or rabbit primary antibody diluted in appropriate antibody diluent (See step 6).
  14. Wash for 5 minutes in buffer.
  15. ImmPRESS™ Reagent. Incubate sections for 30 minutes with appropriate ImmPRESS™ Reagent (anti-mouse Ig, anti-rabbit Ig, or universal).
  16. Wash for 5 minutes in buffer.
  17. Substrate. Incubate sections in second, contrasting peroxidase substrate solution until optimal color develops.
  18. Wash for 5 minutes in buffer.
  19. Counterstain, clear, and mount in appropriate mounting medium.
Add First Primary Antibody