Tissue chip to do multiple immunofluorescence, continuous section thickness and repair conditions how to determine?

The effect of the number of consecutive slices is not as large as the storage time and storage conditions. Generally, TMA wax blocks can be cut 20-50 sheets in a row, but whether the antigen will be significantly weakened depends on the target and the old and new wax blocks. It is recommended to cut out the required number of sheets in succession, dry them in portions, keep them away from light, and complete the dyeing within 1-2 weeks; do not cut dozens of sheets at a time for a long time. You can do a small pre-test: take the 1, 10, and 20 sheets of the same index, and compare the signal strength. If the 20th sheet is obviously weak, reduce the single cut batch or fresh cut. Fragmentation and edge curling often precede antigen decay.

The thickness of 3 μm is feasible for multiple staining of tissue chips, but if CD8, PD-L1 and pancytokeratin are planned to be triple, 3 μm may be thin, the signal is weak, or the slice is easy to roll. I will do 4-5μm of continuous slicing, multiple IF co-location is more stable, and it is not easy to slice. Continuous slicing should ensure that the cross-section of each sheet is close, it is best for experienced technicians to cut it once, and the stall water temperature and baking time are uniform. To use 3μm, cationic slides and low-temperature baking sheets are recommended and the signal strength is confirmed after optimization by antibody.

For multiplex IF, it is not recommended to uniformly lower the temperature; heat retrieval conditions should be optimized separately according to the antibody panel. Commonly used EDTA pH 9.0 or Tris-EDTA pH 8.0, 100°C for 15-20 minutes is relatively friendly for most CD8 and PD-L1; pancytokeratin may also require similar conditions. Single-plex gradients can be performed first: EDTA pH 8.0 vs pH 9.0, 95°C for 15, 20, and 30 minutes, to select conditions that accommodate all three while maintaining low background. During sequential staining, ensure that antibody host species do not conflict, or use TSA cyclic labeling; each round of retrieval will affect the subsequent marker, so it is recommended to stain unstable or low-expression targets first.

There is no uniform standard for the storage time of TMA tablets from different suppliers, which is related to the preparation of wax blocks, antigen repair ability and storage humidity. Generally speaking, the cut white tablets can be kept at 4°C for about 1 month, and -20°C can be slightly longer but not recommended for more than 3 months; repeated fetching and humidity changes can easily lead to antigen loss or slice removal. After purchase, first ask the supplier if there is antigen stability data or how long it is recommended to use it after slicing. If the same batch of chips are different suppliers, take a small number of tablets for time gradient pre-dyeing, and decide to use windows to avoid false negatives when dyeing in large batches.