Technical Guide: How to Perform Multiplex Immunohistochemistry on Frozen Sections

Preface

The core technical principle of multiplex fluorescence immunohistochemistry (mIHC) is based on Tyramide Signal Amplification (TSA) technology. TSA is an enzymatic detection method that uses horseradish peroxidase (HRP) for high-density in situ labeling of target proteins or nucleic acids. For multiplex fluorescence staining, HRP conjugated to secondary antibodies (instead of direct fluorophore conjugation) is used to catalyze inactive fluorophores subsequently added to the system. Under the action of HRP and hydrogen peroxide, these fluorophores are activated and covalently coupled to tyrosine residues of adjacent proteins, enabling stable binding between protein samples and fluorophores. Subsequently, microwave treatment is performed to wash away non-covalently bound antibodies in the previous round, while covalently bound fluorophores remain on the samples. A new primary antibody is then added for the second round of incubation, and this process is repeated. After all antibody incubations are completed and fluorophores are bound, the results are finally detected.

Due to the involvement of multiple rounds of microwave treatment, previously only paraffin sections could withstand such processing. Researchers with only frozen samples could only sigh helplessly, as frozen sections cannot tolerate even one round of heat treatment. However, with technological advancements, this challenge is gradually being overcome. To enable more researchers to utilize mIHC technology, Absin has launched Antibody Elution Buffer (Catalog No.: abs994). Now, let's explore how to conduct multiplex staining experiments on frozen sections!

2. Recommended Experimental Protocol

2.1 Sample Preparation

1.       Fixation and Embedding: Obtain fresh tissues, fix them at low temperature with 4% paraformaldehyde, wash three times with PBS buffer for 15 minutes each time; perform sedimentation dehydration with 30% sucrose, and embed with OCT.

2.       Sectioning: Use a cryostat to cut the tissues into 3-5 μm sections (for tissues stored at -80°C, transfer them to a -20°C refrigerator for equilibration for approximately 15 minutes before section preparation), attach them to adhesion slides, and air-dry the sections at room temperature for 30-60 minutes. Immerse the sections in PBS and wash three times for 5 minutes each to remove OCT.

3.       Endogenous Peroxidase Quenching: Add 3% H₂O₂, incubate at room temperature for 10-30 minutes, and wash with PBS for 5 minutes × 3 times.

2.2 Multiplex Staining Experiment

4.       Blocking: Add blocking solution, incubate at room temperature for 30 minutes, and remove the blocking solution.

5.       Primary Antibody Incubation: Discard the blocking solution, add diluted primary antibody working solution, and incubate overnight at 4°C in a dark humid chamber or for 1-2 hours at 37°C (add a small amount of water to the humid chamber to prevent drying during antibody incubation). Wash with PBS for 5 minutes × 3 times.

6.       Secondary Antibody Incubation: Add HRP-conjugated secondary antibody corresponding to the primary antibody to cover the tissue, incubate in the dark at room temperature for 20-50 minutes, and wash with PBS for 5 minutes × 3 times.

7.       Dye Incubation: Add freshly prepared 1×TSA dye working solution (diluted 1:100 with signal amplification buffer), react for 2-15 minutes, and wash with PBS for 5 minutes × 3 times.

8.       Antibody Elution: Add an appropriate amount of Antibody Elution Buffer (abs994) preheated to 37°C until completely dissolved to cover the sample, shake it off after 10 seconds, add the antibody elution buffer again to submerge the entire sample with a slight bulge, and incubate in a 37°C humid chamber for 5-20 minutes (adjust the elution temperature and time according to section thickness and primary antibody type; Elution difficulty: Structural membrane proteins and cytoskeletal proteins ＞ Cytoplasmic proteins ＞ Nuclear proteins). Wash with PBS for 5 minutes × 3 times.

9.       Repeat the process of [1 Blocking → 5 Antibody Elution] until all target markers are stained.

10.    DAPI Staining: Add 1×DAPI working solution to the sample to submerge the sample area, incubate at room temperature for 10 minutes, and wash with PBS for 5 minutes × 3 times.

11.    Mounting: Add anti-fluorescence quenching mounting medium and cover with a coverslip to avoid air bubbles.

12.    Imaging and Data Analysis: Perform scanning imaging and subsequent data analysis.

3. Key Notes

13.    If certain antibodies are difficult to elute, it is recommended to reduce the dilution ratio of the primary antibody or stain this target in the last round.

14.    When using the Antibody Elution Buffer, adjust the incubation time and elution temperature according to actual conditions. Excessively long incubation may damage antigen targets and nuclear morphology.

15.    Please fix the sample before embedding; post-section fixation may not tolerate multiple rounds of labeling and elution.

16.    For frozen sections, control the section thickness to no more than 5 μm; excessively thick samples may affect staining results.

Although the Antibody Elution Buffer solves the problem that frozen sections cannot undergo heat repair, multiple rounds of elution may still cause section detachment. You can use blank slides to perform multiple rounds of antibody elution to determine the tolerance of the slides, thereby deciding the final number of targets to be stained.

Recommended Absin Products for Frozen Section Multiplex IHC

About Absin (A Sub-brand of ANT BIO PTE. LTD.)

As a professional sub-brand of ANT BIO PTE. LTD. focusing on general reagents and kits, Absin is committed to providing high-quality, reliable experimental products and comprehensive technical support for global life science researchers. Our multiplex fluorescence IHC product series, with the core advantage of breaking the species limitation of primary antibodies and supporting simultaneous detection of multiple markers, perfectly solves the technical difficulties of multiplex IHC experiments on frozen sections. We strive to be your trusted partner in advancing scientific research and accelerating experimental progress.