Fast, uniform, deep 3D nuclear staining BEFORE antibody staining

*This is a simple, robust and fast method for achieving uniform 3D nuclear staining in large, centimeter-scale tissues, including human tissues. Perform all steps at room temperature with gentle shaking and protect samples from light.

1. Delipidate the formalin-fixed tissue using the OmniStain protocol, up to the PBSN washing step, and before the OmniStain buffer A pre-incubation.

2. Incubate tissue for 4 hours in:
0.1M HCl + 0.1M sodium sulfobutylether-β-cyclodextrin (SBECD) in 50% DMSO / H2O

3. Prepare the staining solution as follows:
5-10μM of your desired nuclear dye in 0.1M SBECD in 50% DMSO / H2O

4. Replace the pre-incubation solution with the staining solution and stain for at least 12 hours. Ensure an adequate volume of staining solution relative to tissue size.

5. Wash the tissue with 50% DMSO / H2O for 4 hours.

6. Proceed to tissue clearing or OmniStain A pre-incubation.

Notes:
This protocol yields more uniform and deeper nuclear staining than co-incubation of nuclear dye with OmniStain Buffer C in the general OmniStain protocol.
The protocol is compatible with downstream OmniStain with antibodies. However, nuclear staining should be performed before antibody staining.
The acid serves to convert nucleic acids into an inaccessible form (compaction and neutralisation of negative charges on the phosphate backbone), reducing nuclear dye trapping near the surface, and ultimately enhancing their penetration.
Most proteins in the tissue have an isoelectric point ~6. An acidic medium also reduces the net negative charges on tissue protein surfaces, thereby reducing electrostatic drag against penetration for nucleic acid dyes, which are usually positively charged.
The SBECD complexes the nucleic acid dye, further reducing their trapping by surface-located nuclei, boosting penetration depth and uniformity.