Visikol® HISTO™ Immunolabeling Protocol


Materials That You Will Need

  • Primary and secondary antibodies
  • Nuclear stain – Any common nuclear stain can be used, e.g. DAPI, Hoeschst 33342, SYTOX stains (ThermoFisher Cat# S7020) or TO-PRO-3 (ThermoFisher Cat#T3605)
  • Methanol
  • Phosphate buffered saline (PBS)
  • PBS with 0.2% - 1% Triton™ X-100
  • Container for sample – We suggest glass or polypropylene scintillation vials, Falcon tubes or Eppendorf tube
  • 4% Paraformaldehyde solution, freshly prepared (PFA)
  • Penetration Buffer (PBS / 0.2% Triton™ X-100 / 0.3 M glycine / 20% DMSO)
  • Washing Buffer (Phosphate buffered saline with 0.2% Tween-20 and 10µg/mL heparin)
  • Blocking Buffer (PBS/0.2% triton/6% donkey serum/10% DMSO)
  • Antibody Buffer (PBS/0.2% Tween 20/Heparin/3% donkey serum/5% DMSO) 

Protocol for Labeling and Clearing Tissue

Visikol® HISTO™ has been designed as an easy-to-use tissue clearing and labeling technique for 3D histology. This approach has been adapted from Renier, et al., Cell, 159(4), 896-910.

Email us at info@visikol.com with any questions

1.    Obtain tissues of interest.

Note: Visikol® HISTO™ is effective at clearing unfixed tissues, tissues fixed with a variety of fixatives, as well as clearing over-fixed tissues which have been stored in formalin for years. Tissues are typically fixed via immersion in PFA, but larger tissues, larger than 5 mm (e.g. whole brains) should be perfused with ice-cold 4% paraformaldehyde, or if not possible, slice several channels into tissue (“bread-loafing”) to allow penetration of fixative to avoid autolysis from incomplete fixation of center portion of tissues. Tissues should be placed in a container where the tissue is completely submerged in solution, containing approximately 10x volume of tissue of fixative. We suggest that large tissues like whole mouse or rat brains should be perfusion fixed, as immersion fixation of large tissues can lead to incomplete fixation, autolysis, and necrosis.

2.    Transfer tissues to PBS solution for at least 1 hour twice before further procedures.

Note: Approx. 7 mL of each solution is required for a whole mouse brain whereas 100 µL of each solution is required for a micro-tissue in a well plate. Approx. 3.5 mL and 50 µL should be used with antibody solutions.

3. (Optional for most tissues) Tissues that are particularly difficult to clear due to pigment, collagen, or blood (e.g. liver tissue, whole kidney, over-fixed human tissues), it is recommended to incubate tissue in Tissue Permeabilization Buffer at 37°C with gentle shaking overnight prior to following steps. 

 4. Pre-treatment permeabilization step: Wash tissues in PBS for 5-60 minutes twice, then in 50% methanol (in PBS) for 5-60 mins, 80% methanol for 5-60 mins, and finally 100% methanol for 5-60 mins twice. Samples can be stored in methanol (preferably at 4ºC) indefinitely until ready for next step.

Note: Incubation times for steps will vary with size and nature of sample. Whole mouse brain: 60 minutes; whole rat brain: 60 minutes; Mouse brain hemisphere: 30 minutes; 1 mm mouse brain slice: 15 min; Mouse kidney: 60 minutes; Mouse liver lobe: 2 hours; 3 mm fixed human placenta: 20 minutes; Micro-tissue/organoid (approx. 300 µm diameter): 5 min.

 5. (Optional) For samples which contain substantial quantities of blood or pigment, such as non-perfused heart, lung, kidney, or liver tissue, we recommend bleaching samples by submerging in ice-cold 5% H2O2 in 20% DMSO/methanol (1 part 30% H2O2, 1 part DMSO, 4 parts methanol) and leaving at 4°C overnight. This step will significantly reduce background fluorescence caused by hemoglobin.

6. Wash samples in 20% DMSO/methanol for 5-60 min twice, then in 80% methanol (in PBS) for 5-60 min, 50% methanol (in PBS) for 5-60 min, PBS for 10-60 min twice, and finally in PBS/0.2% Triton™ X-100 for 5-60 min twice before further staining procedures.

Note: Incubation times for each step will vary with size and nature of sample. Whole mouse brain: 60 minutes; whole rat brain: 60 minutes; Mouse brain hemisphere: 30 minutes; 1 mm mouse brain slice: 15 min; Mouse kidney: 60 minutes; Mouse liver lobe: 2 hours; 3 mm fixed human placenta: 20 minutes; Micro-tissue/organoid (approx. 300 µm diameter): 5 min per step.

 7. Incubate samples in Penetration Buffer (PBS / 0.2% Triton™ X-100 / 0.3M glycine / 20% DMSO) at 37ºC overnight.

Note: Incubation times for each step will vary with size and nature of sample. Whole mouse brain: overnight incubation; whole rat brain: overnight; Mouse brain hemisphere: 6 hours; 1 mm mouse brain slice: 2 hours; Mouse kidney: overnight; Mouse liver lobe: overnight; 3 mm fixed human placenta: 1 hour; Micro-tissue/organoid (approx. 300 µm diameter): 30 minutes.

8. For immunolabeling, follow steps 8-11. For other fluorescent probes (e.g. Fluorescent-dextran perfused in vasculature, NeuroTrace™ fluorescent Nissl stain), proceed directly to step 12.

9. Block samples in Blocking Buffer (PBS/0.2% triton/6% donkey serum/10% DMSO) at 37ºC.

Note: Incubation times for each step will vary with size and nature of sample. Whole mouse brain: 72 hours; whole rat brain: 96 hours; Mouse brain hemisphere: 48-72 hours; 1 mm mouse brain slice: overnight; Mouse kidney: 96 hours; E10.5 mouse embryo: 24 hours; E16 mouse embryo: 72 hours; 3 mm fixed human placenta: overnight; Micro-tissue/organoid (approx. 300 µm diameter): 2 hours.

10. Prepare Washing Buffer (Phosphate buffered saline with 0.2% Tween-20 and 10µg/mL heparin); Wash samples with Washing Buffer for 1 hr twice. Transfer samples to primary antibody dilutions prepared in in Antibody Buffer (PBS/0.2% Tween 20/Heparin/3% donkey serum/5% DMSO) and place at 37°C. For most broadly expressing epitopes, a dilution of 1:50 to 1:500 is typically required. Approx. half as much Antibody Buffer should be used as all other solutions to maximize antibody concentration. Incubate samples for the same length of time as they were incubated for Step 8.

11. Wash samples in 1x Washing Buffer (10 times, 5-60 min each time).

Note: Incubation times for each step will vary with size and nature of sample. Whole mouse brain: 60 minutes; whole rat brain: 60 minutes; Mouse brain hemisphere: 30 minutes; 1 mm mouse brain slice: 15 min; Mouse kidney: 60 minutes; Mouse liver lobe: 2 hours; 3 mm fixed human placenta: 20 minutes; Micro-tissue/organoid (approx. 300 µm diameter): 5 min per step.

 12. If using secondary antibody detection, incubate in secondary antibody dilutions (1:50 to 1:500 depending on content of antigen in tissue) in Antibody Buffer at 37ºC. Approx. half as much Antibody Buffer should be used as all other solutions to maximize antibody concentration. Incubate samples for the same length of time as they were incubated in Step 9.

13. (Optional) Add nuclear stain (DAPI, Hoescht 33342, Propidium iodide, TO-PRO-1, etc.) or other stain to a dilution of 1:500 – 1:5000 (depending on stain) in Washing Buffer. Incubate for at least 1 hour.  This step can be done concurrent with antibody labeling steps.

14. Wash in Washing Buffer (10 times, 5-60 min each time) before following steps.  Samples may be kept in this solution indefinitely before further steps.

Note: Incubation times for each step will vary with size and nature of sample. Whole mouse brain: 60 minutes; whole rat brain: 60 minutes; Mouse brain hemisphere: 30 minutes; 1 mm mouse brain slice: 15 min; Mouse kidney: 60 minutes; Mouse liver lobe: 2 hours; 3 mm fixed human placenta: 20 minutes; Micro-tissue/organoid (approx. 300 µm diameter): 5 min per step. Samples which have not been stained with antibodies should require only 3 washes.

15. Treat with 50% methanol in PBS, 3 times at 37°C. Treat with 80% methanol in PBS (filter any precipitated phosphate salts prior to use), 3 times at 37°C. Treat with 100% methanol, 3 times at 37°C.

Note: Incubation times for each step will vary with size and nature of sample. Whole mouse brain: 30 minutes; whole rat brain: 60 minutes; Mouse brain hemisphere: 20 minutes; 1 mm mouse brain slice: 15 min; Mouse kidney: 60 minutes; Mouse liver lobe: 1 hour; 3 mm fixed human placenta: 15 minutes; Micro-tissue/organoid (approx. 300 µm diameter): 5 min per step.

16. Remove from methanol, make sure excess methanol is absorbed with Kim wipe or paper towel.

17. Add Visikol® HISTO-1™ to completely cover the sample – Approx. 7 mL required for whole mouse brain. Clearing time will vary with tissue sample size, but may be accelerated substantially at 37°C without damage to tissue. Additionally, tissues should be agitated gently to accelerate the tissue clearing process. Micro-tissues and thin tissue sections will be cleared with Visikol® HISTO-1™ and can be imaged in this solution, but larger tissues will need to continue to Visikol® HISTO-2™.

Note: Incubation times will vary with size and nature of sample. Whole mouse brain: 24 hours; whole rat brain: 36 hours; Mouse brain hemisphere: overnight; 1 mm mouse brain slice: 3 hours; Mouse kidney: 48 hours; Mouse liver lobe: 24 hours; 3 mm fixed human placenta: 2 hours; Micro-tissue/organoid (approx. 300 µm diameter): 1-2 hours.

18. Larger or thicker tissues will need to be transferred to Visikol® HISTO-2™ to finish the clearing process. Transfer samples and cover (approximately 7 mL for whole mouse brain). Tissues should again be agitated to accelerate the clearing process. Larger tissue samples should be imaged in Visikol® HISTO-2™ solution.

Note: Incubation times will vary with size and nature of sample. Whole mouse brain: 24 hours; whole rat brain: 36 hours; Mouse brain hemisphere: overnight; 1 mm mouse brain slice: 3 hours; Mouse kidney: 48 hours; Mouse liver lobe: 24 hours; 3 mm fixed human placenta: 2 hours; Micro-tissue/organoid (approx. 300 µm diameter): 1-2 hours.

19. Imaging can be conducted using confocal, light sheet or 2-photon microscopy.

20. After 3D imaging tissue clearing can be reversed and the tissue can be processed for 2D histology. See protocol below.


For any questions please email: info@visikol.com


Expected Results


Cleared mouse brain stained with DAPI

Figure 1. Cleared mouse brain tissue, stained with DAPI, single section 500 µm into brain tissue; imaged on Leica TCS SPII Confocal Microscope.

Figure 2. Cleared mouse brain tissue, image taken at 500 um depth into brain tissue, stained with NeuroTrace Yellow (ThermoFisher Scientific, cat #N21481); Imaged with Leica TCS SPII confocal microscope.

Figure 3. HepG2 micro-tissue, cleared with Visikol; labeled for EGFR using rabbit anti-EGFR with anti-rabbit AlexaFluor488-conjugated secondary antibody; z-projection of 10 µm interval optical sections.


References

Renier, N., Wu, Z., Simon, D. J., Yang, J., Ariel, P., & Tessier-Lavigne, M. (2014). iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell, 159(4), 896–910. http://doi.org/10.1016/j.cell.2014.10.010