Thank you for your answer, Mohamed.

It is very difficult for me to approach the frozen tissue on the stage from left side when I take the tissue with brush to transfer it to slide glass. This is because anti-roll plate is on the left side of the stage. 

Now I transfer tissues from the cryostat stage to slide glass using forceps. However, when I put tissues on slide glass, I often get a bubble between the tissue and the slide glass and some folds in tissues.

As for rotating the hand-wheel with my right hand, I don't have any problem.

Do you have any idea to solve my problem? I have tried many methods solve my problem but I still have many bubbles and folds..

Hello, Mohamed san.

Previously I transferred my tissue to cold slide glass and warm up the slides by touching the slide glass from backside.with my finger. However, recently I put the frozen section on slide glass of room temperature. I think the latter method is slightly better but I still get some folds and bubbles.

Dear Yuki,

I'm also a left handed person but I feel that it is an advantage when working with the cryostat. My advice to you is to get all the tools that you need for doing cryosections arranged properly. For example, I'll arrange the brush needed to pick the ribbon inside the chamber. The slides will be readily arranged on the glass covering the cryostat chamber so that it is warm to the room temperature. Then work your way to rotate the wheel with your right hand. As you do this, keep your left hand ready with the brush inside the chamber so that once you start to make your ribbon, you can slowly guide & flatten your ribbon on the cold plate. Once ribbon is ready, use your right hand to pick the glass slide, put down the brush you are holding with the left hand, and with both hands guide the glass slide to pick up the section. One more thing is that you need to properly adjust the temperature for both the chamber and sample holding block. I will usually set-up my chamber to be slightly warmer than the sample holding block so that the sections doesn't curl as much.

It needs a bit of training but so far I've trained a number of students and staff over here in our lab and please feel free to come to our lab if you need some training. Good luck!

Dear Yuki,

I am left-handed and have found that transferring slices into wells filled with PBS/PBS + sodium azide with a paintbrush works quite well (you can then use a paintbrush to arrange the slice on the slide and the liquid makes the tissue more malleable).  If you add a detergent like Triton-X for a rinse after slicing, it will relieve surface tension, decreasing the risk of damaging your tissue while adjusting it on the slide.  

Cheers,

Candace

 Dear Azlin,

Thank you so much for your advice.

In the case of cryostat of my lab, I cannot adjust the temperature of the chamber and sample holding block separately. What is the purpose of setting the chamber temperature slightly higher than sample block?

Yuki 

 Dear Dr. Candace,

Thank you very much for your comment. It's very interesting.

Would you mind if I ask you about  the animal you are working on (human? rodents? fish?) and thickness of the slice you made in cryostat? I am using fish brain (smaller and more fragile than rodent brains) and the thickness of my slice is 20 μm.

I have another question. How do you transfer your brain slices from PBS to slide glass? Do you put the slide glass in PBS?

Yuki

Dear Yuki,

This is to avoid the sections from curling. Proper adjustment will make it easier to get a flatter section. 

Dear Azlin,

Thank you for reply.

Today I have tried to adjust the temperature so that I could get flatter sections. Thank you for good advice.

Yuki

Hi Yuki,

I use rodent brain tissue - adult and neonatal (mouse and rat) and have sliced anywhere from 5-50 µm with a cryostat.  I use a single paintbrush with PBS + TX to transfer the brains from the wells to the slide (I also stain directly in the well which reduces shrinkage of the tissue).  The tissue is much more malleable when it is submerged in the liquid and detergent which allows for manipulation of the sample on the slide.  I have found that a soft bristle brush works best (the tissue tends to stick to stiffer bristles, which can also tear the tissue as well).  Please let me know if you have any other questions.

Cheers,

Candace