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OverviewpdfSDSpdfProtocol


Example protocol


AT A GLANCE

Protocol summary

  1. Fix/permeabilize/block cells or tissue
  2. Add primary antibody in blocking buffer
  3. Add HRP-conjugated secondary antibody
  4. Prepare tyramide working solution and apply in cells or tissue for 5-10 minutes at room temperature

Cat. #

Product Name

Unit

Ex (nm)

Em (nm)

11070

AF 488 Tyramide reagent

200 slides

491

518

11075

AF 546 Tyramide reagent

200 slides

554

570

11082

AF 594 Tyramide reagent

200 slides

590

617

11061

Azido-Cy5 Tyramide

1 mg

644

665

11065

Cy3 Tyramide

1 mg

555

565

11066

Cy5 Tyramide

1 mg

644

665

45100

iFluorTM 488 Tyramide

200 slides

491

514

45105

iFluorTM 555 Tyramide

200 slides

552

567

45110

iFluorTM 647 Tyramide

200 slides

649

665

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Tyramide stock solution (200X):
Add 100 µL DMSO to vial and mix well. Note: Unused Tyramide stock solution can be stored at 2-8o C.

PREPARATION OF WORKING SOLUTION

Tyramide working solution (1X):
Add 100 µL of Tyramide stock solution into 20 mL of buffer of your choice containing 0.003% H2O2Note: Tris Buffer, pH=7.4 can be used for optimal performance. Note: Tyramide working solution should be used immediately and made fresh on the day of use. Note: 20 mL solution is good for 200 tests.

SAMPLE EXPERIMENTAL PROTOCOL

This protocol is applicable for both cells and tissues staining.

Cell fixation and permeabilization

  1. Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.

  2. Rinse the cells or tissue with PBS twice.

  3. Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.

  4. Rinse the cells or tissue with PBS twice.

 Tissue fixation, deparaffinization and rehydration

Deparaffinize and dehydrate the tissue according to the standard IHC protocols. Perform antigen retrieval with preferred specific solution/protocol as needed.

Protocol can be found at
https://www.aatbio.com/resources/guides/paraffin-embedded-tissue-immunohistochemistry-protocol.html

 Peroxidase labeling

  1. Optional: Quench endogenous peroxidase activity by incubating cell or tissue sample in peroxidase quenching solution (such as 3% hydrogen peroxide) for 10 minutes. Rinse with PBS twice at room temperature.

  2. Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.

  3. Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4°C.

  4. Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at 4°C.

  5. Wash with PBS three times for 5 minutes each.

  6. Apply 100 µL of secondary antibody-HRP working solution to each sample and incubate for 60 minutes at room temperature. Note: Incubation time and concentration can be varied depending on the signal intensity.

  7. Wash with PBS three times for 5 minutes each.

 Tyramide labeling

  1. Prepare and apply 100 µL of Tyramide working solution to each sample and incubate for 5-10 minutes at room temperature. Note: If you observe non-specific signal, you can shorten the incubation time with Tyramide.  You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use lower concentration of Tyramide in the working solution.

  2. Rinse with PBS three times.

 Counterstain and fluorescence imaging

  1. Counterstain the cell or tissue samples as needed. AAT provides a series of nucleus counterstain reagents as listed in Table 1. Follow the instruction provided with the reagents.

  2. Mount the coverslip using a mounting medium with anti-fading properties.

  3. Use the appropriate filter set to visualize the signal from the Tyramide labeling.

Table 1. Products recommended for nucleus counterstain.

Cat#

Product Name

Ex/Em (nm)

17548

Nuclear Blue™ DCS1

350/461

17550

Nuclear Green™ DCS1

503/526

17551

Nuclear Orange™ DCS1

528/576

17552

Nuclear Red™ DCS1

642/660

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