Technical Guide: How to Perform Multiplex Immunohistochemistry on Cell Samples

"Hello, my samples are cultured cells. Can I still perform multiplex immunohistochemistry (mIHC)?"

The answer is YES! Multiplex fluorescence immunohistochemistry (mIHC) is compatible with almost all sample types, but it faces a key challenge for cell samples—multiple rounds of antibody elution. Traditional tissue samples, after fixation, embedding, and supported by adhesion slides, have a relatively low risk of detachment during elution. Even if minor detachment occurs, the overall impact is negligible. In contrast, cell samples often have a limited number of cells per slide; any detachment may lead to complete loss of samples. Today, Absin shares a detailed protocol for performing multiplex IHC on cell samples, with our "star product"—Antibody Elution Buffer (Catalog No.: abs994)—playing an indispensable role.

Recommended Experimental Protocol

1.       Sample Preparation: Prepare cell samples (e.g., cell climbing slices, cell smears). It is recommended to use chamber slides for cell culture to facilitate subsequent detection. After preparation, briefly rinse with PBS (2×2 min).

2.       Cell Fixation: Fix cells with 4% paraformaldehyde at room temperature for 10-20 min, then wash with PBS (3×5 min).

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

4.       Cell Permeabilization (Omit for Membrane Markers): Add PBS solution containing 0.1-0.25% Triton X-100, treat at room temperature for 10 min, then wash with PBS (3×5 min).

5.       Blocking: Add blocking solution (5% serum of the same species as the secondary antibody or BSA), incubate at room temperature for 30 min.

6.       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 h at 37°C (add a small amount of water to the humid chamber to prevent drying during incubation). Rinse with PBS (3×5 min).

7.       Secondary Antibody Incubation: Add HRP-conjugated secondary antibody corresponding to the primary antibody to cover the sample, incubate in the dark at room temperature for 20-50 min, then rinse with PBS (3×5 min).

8.       Dye Incubation: Add freshly prepared 1×TSA dye working solution (diluted 1:100 with signal amplification buffer), react for 1-10 min, and rinse with PBS (3×5 min).

9.       Antibody Elution: Preheat the Antibody Elution Buffer to 37°C. After removing PBS, add the elution buffer to cover the entire climbing slice. The elution time can be controlled at 15-30 min (Elution difficulty: Structural membrane proteins and cytoskeletal proteins ＞ Cytoplasmic proteins ＞ Nuclear proteins). Rinse with PBS (3×5 min).

10.    Repeat Staining: Repeat steps [5→9] until all target markers are stained.

11.    DAPI Staining: Add 1×DAPI working solution to cover the sample area, incubate at room temperature for 10 min, then rinse with PBS (3×5 min).

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

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

Key Notes

•         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.

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

•         If both membrane markers and intracellular markers need to be detected, stain membrane markers first, then intracellular markers. Perform permeabilization again before staining intracellular markers; early permeabilization may affect the staining of membrane markers.

•         Due to the potential detachment issue of cell samples, simulate multiple rounds of antibody elution before the formal multiplex experiment. Elute repeatedly with Antibody Elution Buffer and rinse with PBS, then observe cell detachment. If observation under bright field is inconvenient, stain with DAPI for observation.

•         The above protocol may not be suitable for all samples and antibodies; adjust experimental details according to actual conditions.

Recommended Absin Products for Cell Sample 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 species limitations of primary antibodies and supporting multi-marker simultaneous detection, perfectly solves the technical difficulties of cell sample multiplex IHC experiments. We strive to be your trusted partner in advancing scientific research and accelerating experimental progress.