For multiplex immunohistochemistry using pathology diagnostic chips on FFPE samples, the background is high. How can it be optimized?

Blocking and antibody dilution have a major impact on background. For the blocking solution, it is recommended to use 5-10% normal serum from the same species as the secondary antibody for 30-60 minutes at room temperature, or use a protein-free blocking solution, avoiding the combined use of casein/biotin-containing blockers that can cause cross-reactivity. For the primary antibody diluent, PBS containing 0.1-0.3% Triton X-100 and 1% BSA can be used. If the background is too high, the primary antibody concentration can be reduced 2-4 fold and the 4°C incubation time extended; the secondary antibody concentration can also be appropriately lowered. Add 0.05% Tween-20 to the wash buffer, 5 minutes per wash × at least 3 washes. For multiplex fluorescence, special attention must be paid to secondary antibody species cross-reactivity and dye cross-talk.

Pay attention to the sequence and signal amplification in multiplex IHC. First, perform single staining for each marker separately to confirm the positive intensity and background of each antibody on old samples; when combining multiple markers, start staining with the markers that have weak signals and fragile antigens to avoid damage from repeated elution. If the fluorescence is weak, consider tyramide signal amplification (TSA), but TSA will amplify background, so preliminary blocking and H2O2 quenching must be sufficient, and single-staining controls must be set up to rule out cross-reactivity. It is recommended to first test a small area or a local region of the entire slide, using a PAP pen to separate different conditions, to reduce sample consumption.

For old FFPE samples with high background and weak signal, start with antigen retrieval first. It is recommended to use pH 9.0 EDTA or Tris-EDTA with pressure cooker retrieval, and the time can be extended by 5-10 minutes compared with the routine protocol, but avoid drying the sections; after retrieval, allow natural cooling at room temperature for 20-30 minutes before opening the lid. For endogenous peroxidase blocking, use 3% H2O2 for 15 minutes; for fluorescence detection, autofluorescence also needs to be considered. After retrieval, first use DAB single staining for CK or MLH1 to verify staining intensity, and do not start with multiplex labeling right away. For dozens of samples, it is worth using 2-3 slides to run a retrieval condition gradient, identify the optimal point, and then process all samples uniformly.

When the sample size is small, do not run trial-and-error across the entire batch. You can take 1-2 sections and use a PAP pen to divide them into 4-6 small areas, then set up a small matrix of antigen retrieval conditions or blocking conditions, staining only one marker per grid. High background may also be due to autofluorescence from old paraffin blocks, which can be treated with 0.1-0.5% Sudan Black B or a commercial autofluorescence quenching reagent; this is quite helpful for multiplex fluorescence. Record the retrieval temperature, blocking solution lot number, and antibody dilution ratio for each round, and keep scanned images of the same slide for comparison. Contact the antibody manufacturer's technical support for recommended concentrations and diluents; sometimes there are specific optimizations for FFPE.