whenever I take cryostat sections of rat brain, many holes are distorting the normal morphology(image attached). what may be the reason?
protocol which we follow:
* Perfuse the brain in PBS and then store in 4% PFA overnight (4 deg).
*Transfer and keep the brain in 15% sucrose until it sinks(4 deg).
*Transfer and keep the brain in 30% sucrose until it sinks(4 deg).
*Take out the brain and wipe the excess sucrose using tissue paper, and keep it in cryostat set to -20 °c.
*Embed the tissue using OCT media on a metal chuck by covering the tissue layer by layer inside the cryostat.
*Start sectioning.
Anirban Barik The presence of holes in cryostat sections of rat brain tissue can be due to several factors. Here are some common causes and potential solutions:
Here are the protocol that we follow. You can choose either based on your feasibilty.
Protocol for Preparing and Sectioning Rat Brain Tissue Using Cryostat
Protocol 1: Direct Freezing and Sectioning
Prepare Isopentane for Freezing
Place a small amount of liquid nitrogen in an icebox.
Place a 100-200 ml beaker into the liquid nitrogen, ensuring the liquid nitrogen level is higher than the isopentane level in the beaker.
Add isopentane to the beaker
Allow the isopentane to freeze until a whitish line forms around the beaker. Scratch this line to check if it is ready. Jelly-like isopentane should form. If it completely freezes, take out the beaker and place it at room temperature until it reaches a jelly-like consistency.
This preparation should be done just before you take the brain after perfusion.
Freeze the Brain:
Using a spatula, dip the brain into the jelly-like isopentane for 10 to 20 seconds. Avoid touching the walls of the beaker to prevent damaging the brain surface.
After freezing, place the brain into a tube or bottle and store it at -80°C overnight for rapid freezing.
Cryostat Preparation:
Bring the brain to the cryostat station on liquid nitrogen.
Turn on the cryostat and set the temperature to around -23°C to -26°C. Place all items (e.g., brushes) into the cryostat chamber.
Keep the lid closed to stabilize the temperature.
Acclimate the Brain:
Place the brain in the cryostat for 10–20 minutes to acclimate to the cryostat temperature.
Prepare OCT:
Place the dye into the cryostat and wait for 10–20 minutes.
Put 1 to 2 drops of OCT on the dye. If it starts freezing immediately, the temperature is appropriate for embedding the brain.
Embedding and Sectioning:
Place the brain on the dye with a minimal amount of OCT, focusing on the base rather than covering the whole brain.
Adjust the position of the brain as required for cutting.
Use a new, sharp blade for sectioning.
Cut sections at the desired thickness, typically 10–20 µm.
Protocol 2: Embedding in OCT Before Freezing
Freezing: Prepare Isopentane for Freezing
Place a small amount of liquid nitrogen in an icebox.
Place a 100-200 ml beaker into the liquid nitrogen, ensuring the liquid nitrogen level is higher than the isopentane level in the beaker.
Add isopentane to the beaker.
Allow the isopentane to freeze until a whitish line forms around the beaker. Scratch this line to check if it is ready. Jelly-like isopentane should form. If it completely freezes, take out the beaker and place it at room temperature until it reaches a jelly-like consistency.
This preparation should be done just before you take the brain after perfusion.
Freeze the Brain in OCT:
Take an aluminum foil, put OCT onto it, and place the brain into it. Avoid damaging the brain and ensure no air bubbles are trapped in the OCT.
Take a plastic mold, fill it with OCT, and place the brain into the mold.
Take the isopentane solution in a beaker in dry ice, and place the mold in the isopentane for 5-10 minutes, ensuring that the isopentane does not enter the mold.
Once completely frozen, wrap the brain with OCT in aluminum foil and store it at -80°C overnight.
Sectioning: Cryostat Preparation
Bring the brain to the cryostat station on liquid nitrogen.
Turn on the cryostat and set the temperature to around -23°C to -26°C. Place all items (e.g., brushes) into the cryostat chamber.
Keep the lid closed to stabilize the temperature.
Acclimate the Brain:
Place the brain in the cryostat for 10-20 minutes to acclimate to the cryostat temperature.
Prepare OCT:
Place the dye into the cryostat and wait for 10-20 minutes.
Put 1 to 2 drops of OCT on the dye. If it starts freezing immediately, the temperature is appropriate for embedding the brain.
Embedding and Sectioning:
Place the brain on the dye with a minimal amount of OCT, focusing on the base rather than covering the whole brain.
Adjust the position of the brain as required for cutting.
Use a new, sharp blade for sectioning.
Cut sections at the desired thickness, typically 10-20 µm for brain tissue.
Mounting Tissue on Slides:
After sectioning, place the tissue sections onto slides. Allow the slides to sit at room temperature for 30 minutes to adhere properly. Place the slides at -80°C overnight.
Staining:
The next day, remove the slides from the -80°C storage.
Proceed with the staining protocol for your specific application.
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