A Quick Guide to OmniStain

Introduction

• OmniStain (also known as INSIHGT) is a multiplexed 3D tissue immunostaining method.
• Increases antibody penetration depth and uniformity by 20-30x, perfect for high-quality tissue clearing.
• The staining only takes 4 days for whole adult mouse brains, or equivalently sized tissues (~1cm³-sized) and does not require specialized equipment or special tissue pre-treatment. Parallel tissue processing is straightforward using multi-well cell culture plates.
• Fluorophore-conjugated primary antibodies are NOT required. To date, >360 commercially available primary antibodies have been proven to be compatible with INSIHGT and the list continues to grow.
• OmniStain quenches endogenous fluorescence, so the localization of fluorescent protein must be revealed with their corresponding anti/nanobodies.
• Before you start, please read this guide carefully and watch our video demonstration below.

Read the paper: Yau et al. Nature communications. 2024. 15: 10888.

Overview

OmniStain consists of 3 steps. For any formaldehyde-fixed tissues (typically 4% paraformaldehyde-fixed or 10% neutral buffer formalin-fixed), the tissue will go through (1) delipidation, (2) OmniStain staining, and (3) solvent-based tissue clearing, before proceeding to 3D multiplexed imaging.

Materials provided in the kit

• 2x OmniStain buffer A
• 1x OmniStain buffer B
• 1x OmniStain buffer C

OmniStain protocol

** perform all steps at 20-25oC (room temperature) unless otherwise specified.

Delipidation

1. Dehydrate the fixed tissue in 50% v/v Methanol/H₂O, then 100% Methanol x 3, 30 min each.
2. Delipidate the tissue in 2:1 v/v mixture of CH₂Cl₂/Methanol (delipidation mix) overnight in a glass, or polypropylene (PP) container. Mix and add the solutions in a fume hood.
3. Wash the tissue in 100% Methanol x 3, 50% Methanol/H₂O x 2 , and PBSN x 2, 30 min each.
4. Pre-incubate the tissue in 1x OmniStain buffer A at 37°C overnight. Prepare the 1x OmniStain buffer A by mixing 2x OmniStain buffer A with equal volumes of PBSN.
   
   OmniStain staining
   ** note the values were illustrated for 1 cm³ tissues or whole adult mouse brains, and can be scaled accordingly.
5. Remove and discard the spent 1x OmniStain buffer A in pre-incubation.
6. Place the tissue in a tightly fitting container.
   • ** For 1 cm³ tissues / whole mouse brain, use a 5 ml Eppendorf tube or a 15 ml Falcon tube.
7. Add 2x OmniStain buffer A to half-cover the tissue.
   • ** For 1 cm³ tissues / whole mouse brain, use 250 μl 2x OmniStain buffer A.
8. Add primary antibodies or lectins. For 1cm³ tissues / whole mouse brain, start with 10μg each.
   • ** You can add as many species-compatible primary antibodies as you wish for multiplexed 3D IHC.
   • ** You can reduce the amount of primary antibodies used if the staining penetration is satisfactory.
9. Add equal μg amount of fluorescent secondary antibody Fab fragments or nanobodies.
   • ** Fab fragments or nanobodies must be used instead of whole IgG secondary antibodies.
10. (Optional) In a separate tube, premix desired amount of nuclear staining stock solution (regardless of concentration) with equal volumes of 1x OmniStain buffer C. Then add mixture to the staining solution.
11. Top up with PBSN to a final 1x OmniStain buffer A concentration.
    • ** The volume required = (volume of 2x OmniStain buffer A in step 7) – (total volume of solutions added through steps 8-10).
12. Top up with 1x OmniStain buffer A to fully cover the tissue.
    • ** Use a minimal volume of 1x OmniStain buffer A to just cover the tissue.
13. Incubate at room temperature (20-25oC) for 3-7 days(for 1-2cm³ tissues) with gentle shaking in the dark.
    
    Washing and Tissue clearing
14. Wash the tissue in 3-times the tissue volume of 1x OmniStain buffer B at room temperature (20-25oC) for 6 hours. Shake the tube horizontally to ensure adequate washing.
    • ** For 1cm³ tissues / whole mouse brain, use 3 ml 1x OmniStain buffer B.
15. Wash the tissue thoroughly with PBSN x 3 for 2 hours each at room temperature. Shake the tube horizontally to ensure adequate washing.
16. Dehydrate the tissue as in Step 1. Then incubate and clear the tissue in solvent-based RI matching solution. This can be 1:2 v/v mixture of benzyl alcohol / benzyl benzoate (BABB), dibenzyl ether (DBE), or ethyl cinnamate (ECi). Clear the tissue by incubating for 2 x 3 hours each at room temperature.

OmniStain using conventional (whole IgG) fluorophore-conjugated secondary antibodies

It is possible to use the more common fluorophore-conjugated secondary antibodies (whole IgG format) rather than their Fab fragments or nanobodies counterparts. However, the primary antibodies and secondary antibodies must be added separately, meaning the tissue must be stained twice with near doubling of tissue processing time. The penetration homogeneity will also be suboptimal.

• Follow the standard OmniStain protocol as above till Step 8. Then skip Step 9, and proceed accordingly from Step 10 to Step 15.
  • Now you will obtain a tissue stained with primary antibodies.
• Pre-incubate the primary antibody-stained tissue again in 1x OmniStain buffer A as described in Step 4. Then follow through Step 5 to Step 7.
• Skip Step 8 and add conventional fluorescent secondary antibodies (whole IgGs) at half the amount as specified in Step 9.
• Process the fluorescent stained tissue through the rest of the protocol.