Standard Perfusion
Before SHIELD begins, the animal should be perfused to remove residual blood which can cause issues with autofluorescence during imaging or nonspecific antibody labeling.
Reagents/Equipment Required
- 32% Paraformaldehyde Solution: (EMS) 15714-1L
- 1x PBS
- Heparin – Sigma H3149
Optional for decalcification if your sample contains bone:
- Ethylenediaminetetraacetic acid (EDTA) – Sigma E9884
- Sodium hydroxide (NaOH) – Sigma 415413 (or any NaOH pellets)
Protocol
Perfusion and Extraction
1. Transcardially perfuse the animal with ice-cold heparinized PBS. For mice, use about 20 mL and a 5 mL/min flow rate. For rats, use 200 mL and a 60 mL/min flow rate.
We recommend using heparinized PBS to remove as much blood as possible (20 U/mL concentration).
Perfuse with PBS until the fluid is running completely clear of blood before perfusing with ice-cold 4% PFA in PBS. Use the same volume and flow rates as before.
Take extra care not to introduce air bubbles inside tubing.
When the fluid comes out of the mouth or a lung swells, adjust the position of the needle in the heart.
[Note] The better the perfusion, the better the results!
2. Dissect out the brain / organ of interest. Careful dissection is essential to preserve the sample’s structural integrity.
[Note] Try to avoid physically damaging the sample when extracting it. Physical damage can cause issues with potential atlas alignment during analysis, or can serve as a location to nonspecifically trap antibodies during labeling.
3. Incubate the sample in 4% PFA in PBS for 24 hours at 4°C with shaking. If you are not ready to continue to the next step, the sample can be stored in 1X PBS with 0.02% sodium azide at 4°C until you are ready to continue.
Remove Interfering Tissues (If necessary)
This will be needed on samples such as whole mouse heads.
4. For any sample, it is best to remove skin or hair as much as possible — keratin does not clear very well.
5. For whole mouse heads or any samples containing bone, they will need to be decalcified. Soft tissues can skip this step!
- Prepare a solution of 10% EDTA in distilled water. EDTA’s solubility is pH dependent, so add NaOH to dissolve the EDTA and adjust to pH ~7.5.
- Incubate the sample in 10% EDTA at 4°C until the bone is soft and flexible. For a whole mouse head this takes 6 days.
You have now completed standard perfusion. Continue to the SHIELD home page to evaluate SHIELD protocol options: