TITLE: Preparing a 3D Printed Implant for Acute In Vivo Electrophysiology
AUTHORS: Xinxin Yin, Anna Lakunina, Josh Siegle

[DESCRIPTION]
When performing acute electrophysiology experiments in mice, replacing the skull with a 3D printed implant several weeks in advance can substantially improve recording quality. Prior to surgery, these implants must be coated with a layer of silicone to maintain brain health. This protocol describes the steps necessary to apply this silicone layer in advance of a cranial windowing procedure.

[BEFORE_START]
The implants need to be designed and printed in advance, for more details see Bennett et al. (2024) bioRxiv (DOI: 10.1101/2023.11.12.566771):

[STEPS]
SECTION: Coat the implant
1. Prepare the   . Put the solid putty on a flat surface (like the bottom of a glass beaker) and heat the putty in the micro-wave oven. Check the softness of the putty every 30 s to prevent overheating. Stop heating when the putty is hot to the touch and easily malleable.

SECTION: Coat the implant
2. Manipulate the putty to create a flat surface with no creases.
SECTION: Coat the implant
5. 
 
Insert the implants into the indentations. Gently press the implants into place, so the bottom of the implant fully makes contact with the putty. Do not press too hard, as you do not want the putty to fill the holes in the implant.
SECTION: Coat the implant
6. Prepare the   (including Part A and Part B).

SECTION: Coat the implant
6.1. In a beaker, weigh out 20 g of Part A, and 2 g of Part B.

SECTION: Coat the implant
6.2. Mix the two parts together thoroughly to combine them, until the mixture is homogeneous.
SECTION: Coat the implant
6.3. Use a degasser to remove bubbles from the mixture. Keep the mixture in the degassing chamber until you see the mixture rise, then collapse as the bubbles burst. Wait another minute after the collapse before removing the mixture from the degasser.
SECTION: Coat the implant
7. Draw the degassed SORTA-Clear into a 1 mL Luer-lock syringe. Attach a 25G blunt needle.
SECTION: Coat the implant
8. 
 
Cover the top of the implants with SORTA-Clear.
SECTION: Coat the implant
8.1. Place the putty with the implants under a dissection scope to better see what you're doing.
SECTION: Coat the implant
8.2. Fill each hole in the implant with SORTA-Clear using the 25G needle. Take care not to create bubbles by dispensing SORTA-Clear near the hole and allowing it to flow into the hole on its own. If bubbles do form, try to dislodge them with the needle.
SECTION: Coat the implant
8.3. When each hole is filled, cover the entire top surface of the implant (excluding the outer lip) with a layer of SORTA-Clear.
 

SECTION: Coat the implant
9. Allow the SORTA-Clear to fully cure for at least 1440 min  before removing the implants from the putty and implanting them into animals.

SECTION: Coat the implant
8.4. Repeat for each implant. You have about an hour to work with the SORTA-Clear before it cures too much to work with. If it is getting very difficult to push it out of the syringe, make a fresh batch.
SECTION: Sterilizing the implant
10. Coated implants are then cleaned by rinsing in 70% ethanol, placed into individual autoclave bags, and autoclaved for 5 min (131°C, 14.7 psi). Note that autoclaving at higher temperatures can lead to defects in the implant.

SECTION: Coat the implant
4. 
 
Use the 3D-printed stamping tool to create indentations in the putty. One indentation is needed for each implant being prepared.
SECTION: Coat the implant
3. Disinfect the implant by soaking in 70% ethanol for a few seconds.