To provide better resolution and SNR and to avoid motion artifacts

Adapted from Lerch, Sled & Henkelman., (2011) by Ph.D.M.Sc Luis Angel Trujillo Villarreal

Materials

Perfusion solution
PBS 10X
Heparin sodium 5,000 IU/ml
Gadobutrol 1 mmol/mL (604.72 mg/mL)
Distilled water

Fixation solution
PBS 10X
Heparin sodium 5,000 IU/ml
Gadobutrol 1 mmol/mL (604.72 mg/mL)
Distilled water
4% Paraformaldehyde solution

Posfixation solution
PBS 10X
Sodium azide 0.02%
Gadobutrol 1 mmol/mL (604.72 mg/mL)

4% paraformaldehyde solution
Paraformaldehyde powder 95%
Sodium hydroxide concentrate 0.1 M

Anesthetics
Pentobarbital sodium 65 mg/ml

Require lab equipment and materials
Extractor hood
Magnetic Stirrer hot plate
2 Magnetic stirrer
Analytical scale (for small weights < 1 gr)
Digital scale (for weights > 1 grs
3 Beaker or Erlenmeyer matraz of 1 lt (If your volume would be more than 1 lt, use a bigger beaker)
Multiple glass storage containers with lids (Take into account your intended volume of every solution for consider the quantity of your storage containers)and for the fixation solution, use amber glass containers if possible, if not, you could use something to cover like aluminum foil
2 graduated cylinders

It is a good practice to measure every volume separately (in different graduated cylinders or syringes). Be sure to wash all the materials before using to avoid contamination.

Solution preparation

Perfusion solution

200 ml per rat (consider a margin of error of 50 ml per rat)

$$\text{PBS 10X}= \text{(Calculate the 10\% of the intended total volume in ml, that would be how many ml of PBS are needed)}$$

$$\text{Heparin (ml)}= {10\times\text{Total solution volume (ml)}\over 5000\text{(UI/ml)}}$$

$$\text{Gadobutrol (ml)}= {4\mu M\times\text{Total solution volume (ml)}\over 500\mu M}$$

$$\text{+ Distilled water = (The necessary volume to reach the final desired volume after the sum of the previous)}$$

Use a beaker and a magnetic stirrer to make the solution, no heating is needed. Storage in the refrigerator in a storage container with a lid. Filter if needed.

4% Paraformaldehyde solution

1. Weigh the PFA powder first. To find out how much, you must obtain what is the 4% of your total liquid.

$${\text{PFA 4\%}= \text {1000 ml}\times 0.04}$$

2. Add the PFA to distilled water. Under the extraction hood, stir gently on a heating block at ~57° C. Add a lentil of Sodium hydroxide. Stir until the paraformaldehyde is dissolved. You must obtain a clear solution (water clear).

Using sodium hydroxide concentrate 0.1 M solution Based on M.Sc student M.D. Juan Pablo Maya preparation method

For each liter of PFA% add 24 to 30 ml of sodium hydroxide concentrate 0.1 M or 480 to 600 drops. This will clarify the solution. Continue stirring and control the temperature to stay at ~57° C

3. Let it cool until room temperature.

4. Filter the solution if necessary.

Notes: PFA is a chemical highly toxic to skin and mucous membranes, be sure to use adequate protection to handle the powder and the solution.

Fixation solution

First, you must consider the concentration of your PFA 4% solution. If it was intended to be prepared to the fixation solution, your PFA solution must be half of the fixation solution, then add your 4% of powder (calculated by the total volume of the fixation solution) in half of the water volume.

$$\text{Gadobutrol (ml)}= {4\mu M\times\text{Total solution volume (ml)}\over 500\mu M}$$

$$\text{Heparin (ml)}= {10\times\text{Total solution volume (ml)}\over 5000\text{(UI/ml)}}$$

$$\text{PBS 10X}= \text{(Calculate the 10\% of the intended total volume in ml, that would be how many ml of PBS are needed)}$$

Total amount of the solution = (4% Paraformaldehyde solution volume) + Gadobutrol volume + Heparin volume + PBS 10X volume + With Distilled water go to the total amount of solution that you want.

Example:

I need to prepare the fixation solution for 4 rats, then am gonna need 800 ml (200 ml per rat).

Total needed 800 ml

To prepare PFA at 4% use 400 ml of water and (800 X 0.04%) 32 gr. of PFA powder. This will be half of the total amount needed.

$$\text{Gadobutrol (ml)}= {4\mu M\times\text{800 (ml)}\over 500\mu M} = \text {6.4 ml}$$

$$\text{Heparin (ml)}= {10\times\text{800 (ml)}\over 5000\text{(UI/ml)}}= \text {1.6 ml}$$

$$\text {PBS 10X} = \text {80 ml} $$

$$ \text{6.4 ml Gadobutrol + 1.6 ml Heparin + 80 ml PBS = 88 ml to 400 ml, add 320 ml of distilled water}$$

$$ \text{400 ml PFA 8\% + 400 ml solution (gado + hep + PBS + water) = 800 ml fixation solution}$$

***** It is ideal to use your fixation solution in the first 72 hrs because the solution will re-polymerize during storage it is best to use it immediately or within a few days. Storage in a refrigerator (4 Centigrades) until use (Paraformaldehyde, Formaldehyde, and Formalin | Light Microscopy Core Facility, n.d.) *****

Posfixation solution

You'll need this solution to store your samples, so the amount of solution will depend on how many samples you have collected.

1. PBS 10X
2. Sodium azide 0.02%
3. Gadobutrol 1 mmol/ml (604.72 mg/ml) * Add at the end to calculate for any errors, due to the high cost of this solution.

Hypothetically if you are interested in preparing 200 ml of this solution, follow the next:

$$ \text{PBS 10X = 20 ml}$$

$$\text{Sodium azide = 0.04 gr}$$

$$\text{Gadobutrol = 1.6 ml}$$

$$\text{+ Distilled water = 178.36 ml}$$

Consider a margin of error of 50 ml per rat.

Storage in a container with a lid in the refrigerator. Avoid contact with eyes and mucous when manage.

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Transcardial perfusion

Materials
Perfusion pump
Pump hoses
Hypodermic needles 21G with a sanded edge (are sanded by the user)
Dissecting forceps
Fine scissors
Tissue scissors
Two hemostatic forceps
Curette
Timer
Masking tape or micropore

Perfusion pump configuration

To preserve the integrity of the blood vessels, the strength and speed of the solution must be controlled. It is necessary to program the flow rate of 200 ml in 20 minutes, i.e. 10 ml per minute. Be sure to make this programation with your setup ready.

Procedure

Be careful handling the rats, and protect always your hands. Ask for help if needed. If you notice the rat is uneasy, you may want to give'em time to calm down. It will facilitate the anesthesia induction effect.

1. Rodents are anesthetized with pentobarbital sodium 65 mg/ml via intraperitoneal injection. According to Zatroch et al., (2017) a dose of 800 mg/kg is the most effective method for Sprague-Dawley rats because both dose and volume contribute to the speed of death.
2. Wait until profound anesthesia is reached (paralyzed vibrissae, no blinking, no limb movement, no withdrawal to painful stimuli).
3. Place the rat in a supine position in your setup. Locate the anatomical landmark of the xiphoid appendix and do your fist cut orthogonal to it.
4. With the help of the hemostatic forceps take the xiphoid.
5. Continued the cut lateral until reached the anterior axillary line (be careful when puncturing the lungs, make sure that the tips of the scissors are always pointing in the direction of the ribs you are cutting).
6. The continued orthogonal in a cephalic direction until reached an imaginary line between bilateral the 3rd ribs.
7. Expose the thoracic cavity by pulling the hemostatic forceps in the direction of the head. Leave them on the sides of the head.
8. Use the dissection forceps to grab the heart (carefully but surely) with your left hand, and with your right hand grab the needle by the base (remember always look out for your hands). Localize the apex of the heart and punction there until you see the needle goes for 1 cm approximately (this is for rats, for mice it might be lesser).
9. Free your left hand to take the base of the needle with that hand, and now with your right hand secure the needle position with the help of masking tape or micropore.
10. Turn on the perfusion pump (check that the configuration is preserved and that the direction of the flow is correct (pump-body))
11. Start the timer, 200 ml of the perfusion solution should flow in 20 min and 200 ml of the fixation solution in 20 min.
12. Make the necessary adjustments to allow for cleaning, you could reposition the rat, and widen the lateral cut of the ribs to allow fluid to flow and reach the thoracic cavity. It is of utmost importance that you have good visibility of what is going on so that you can correct it in case of any inconvenience.

In this figure the rat is in the supine position and the anatomical landmarks are seen. It also shows where the heart is, and the ways in which the needle should penetrate the myocardium (the marked lines are the possible permissible ways of approaching the left ventricle)

**If you accidentally puncture an organ or the heart in a different place than intended. You could use your other hemostatic forceps and place them just above where you see the injury. The weight of the forceps may help close the wound. **

Skulls

To separate the head from the body, use a guillotine or garden shears.

Avoid using the materials for something for which they are not intended (e.g., scissors to dislocate or cut bones).

Use the curettage to remove the skin and the cartilaginous tip of the nose. Use the fine scissors to remove the lower jaw and ears.

The skulls should remain in the fixation solution for 12 hours and then be transferred to the postfixation solution, where they should remain for at least 2 weeks, as reported by Ph.D.M.Sc Luis Angel Trujillo Villarreal

References

1. Lerch JP, Sled JG, Henkelman RM. MRI phenotyping of genetically altered mice. Methods Mol Biol. (2011) ;711:349-61. http://doi.org/10.1007/978-1-61737-992-5_17. PMID: 21279611.

2. Paraformaldehyde, Formadehyde and Formalin | Light Microscopy Core Facility. (n.d.). Retrieved October 26, 2022, from https://microscopy.duke.edu/guides/paraformaldehyde-formaldehyde-formalin

3. Zatroch, K.K., Knight, C.G., Reimer, J.N. et al. Refinement of intraperitoneal injection of sodium pentobarbital for euthanasia in laboratory rats (Rattus norvegicus). BMC Vet Res 13, 60 (2016). https://doi.org/10.1186/s12917-017-0982-y