- Anesthetic (ketamine 50 mg/Kg and xylazine 4.5 mg/Kg – varies according to institutional protocols)
- Peristaltic pump (Gilson) with tubing and connectors
- Dissection tools (scissors, fine scissors, spring scissors, tweezers, spatula, according to preferences)
- PFA stock solution (recommended: 16% PFA solution, Electron Microscopy Science)
- pHmeter and related reagents/tools
- Perfusion needle (preferred: 27 gauge ½ inch)
- Solid PFA waste collection bin
- Liquid PFA waste collection bin
- Vacuum-trap jar and connector tubing
- Performing the procedure and handling PFA under a fume hood is strongly recommended.
- Lab gown/disposable gown
- Examination gloves
(cut-resistant gloves are recommended)
- PBS can be prepared from 10X concentrated solution
- 4% PFA solution is prepared by diluting the concentrated PFA stock and PBS 10X stock in water. For better results, it is recommended to prepare a fresh 4% PFA solution in PBS right before the procedure. Adjust pH of PBS and PFA solutions to 7.3-7.4
- Vibratome (VTS1200S Leica microsystems) with removable tray, cutting chamber and vibro-check tool.
- Dissection tools (tweezers, spatula, according to preferences)
- Double-edged razor blades
- Single-edge razor blades
- Medium weighing bowl or petridish
- Pre-solidified 2% agarose
- Plastic or glass transfer pipette/small brush
- 6/12/24 wells cell culture plates (according to preference) to collect slices
- Lab gown/disposable gown
- Examination gloves (cut-resistant gloves are
recommended)
- Blocking reagent, e.g.: Normal Goat Serum (NGS) – the correct blocking reagent/blocking solution should be established based on the characteristics of the antibodies used.
- Triton-X100 (detergent)
- Hard-drying mounting medium (recommended ProLong Diamond, ThermoFisher Scientific)
- Glass coverslips (recommended #1.5, VWR)
- Lab coat/disposable gown
- Pre-mounted microscopy slides
- Confocal laser scanning microscope with appropriate objectives (recommended: 10x/0.4 or a 60x/1.35 immersion) connected to a computer with the appropriate imaging software
- 70% Ethanol/lens cleaning solution
- Image processing software (recommended: FIJI).