So adding mounting medium and then placing the coverslip onto the slide sounds and looks easy enough, but there's still so many things that could go wrong. For one, I just can't seem to be able to completely avoid the air bubbles which is really frustrating when your trying to get a good image and there's a small bubble right in the area of interest. Does anyone have some good advice for this last step in the IHC process? All kinds of tips and tricks are welcome. My spinal cord tissue are mounted on slides and my brain sections are free-floating, but both seem to have the same with air bubbles.
I always centrifuge the mounting medium for 12000rpm/20min at 4 degree first. Then gently use the pipette to drop the mounting medium on one side of the slide. Then gently and slowly, particularly for brain with ventricle spaces, slide down the coverslip. For regular IHC, seal the slide afterward and you will see no bubbles at all. But for IF, sometimes, even after I seal the slide, maybe after 30min, I can still see tiny bubbles under the microscopy. So just image your slides ASAP. Hope it helps!
When I drop the mounting medium on the tissue/slide, I usually do not use the first drop as there maybe some bubbles in it. Another tip: when I put the coverslip on the the slide, I usually hold the coverslip at 45 degree and let one side of coverslip attached the slide. And then I slowly release the coverslip to attach to the slide, the bubble in the mounting medium will go with coverslip to the other side of it. The bubbles will be easily to remove after you slightly push the coverslip. Hope it helps!
I usually drop on more of the mounting medium than needed and then gently suck some back up with the pipette whilst trying to suck up any bubbles I can see. Of course be careful not to suck up or move the slice. As mentioned I also put the cover slip at a 45 degree angle and let it slowly drop down from one side to the other. I find using a paintbrush to slowly lower the cover slip is a good way to keep control. Hope this helps!
you want to put the sample on the slide, carefully soak up the liquid around it with a kimwipe and then let it sit for a few minutes to dry before putting the mounting medium on. If it isnt dry enough the sample will move around when you put the mounting medium on and that could cause problems when putting the cover slip on.
- Badges
- Science topic
- Similar topics
- Computer Science
- Images