Poor PFA fixation often causes trouble in antibody staining. Folded cells, poorly preserved cytoskeleton… These artifacts appear when the stock of PFA gets older and degrades. Buying ready made PFA solutions, most of which contain 10-15% of methanol, can also lead to low labelling with some antibodies.
Glyoxal seems to be a good alternative. It had the added advantage that it is less toxic.
Check this article to know more. 🙂
The Live Cell Imaging facility is back with its intensive microscopy course! In 2018 we moved to a new building so there was no course but we will strike again in Jan-Feb 2019!!You will definitely learn tons at our course. Have a look at the program and judge for yourself.
As usual we run 2 courses in parallel:
#2870, 6 points, is the full course with all lectures, workshops, demos… This will run 22/jan-08 feb, 3 days/week 9:00-17:15.
#2871, 4.5 points, is the same minus some workshops and demos. This course will run at the same dates but 10:00-15:00.
The rest of the time is used for home assignments.
All lectures are open to the public without any registration so tell your colleagues!
Course applications are open from today and until the 15th of November. The full course (2870) has only 16 spots available so just go for it NOW! 🙂
As you all (nearlyish) know, one should never place a sample on a thick glass slide and add a coverslip. Instead, the sample should be placed on the coverslip then covered with a thick glass slide. And the coverslip should be 170 um thick (also labelled thickness #1.5).
Why is that? Because the coverslip is part of the design of the objective and all objectives from all manufacturers are designed to image through 170 um glass and assuming that the sample is directly in contact with the coverslip.
What about superfrost slides that one uses to make sure tissue sections don’t float away during antigen retrieval? No worry! You can make your own superfrost coverslips. It is cheap and you can prepare tons at the same time. Here is the protocol.
Not convinced? You will only see the difference when you compare side by side! The images of your tissue will be much sharper if the sample is on the coverslip because when you put your sample on the slide, both the thick glass and the mounting medium end up between the objective and the sample. The objective is not designed for this. 😀
Have you ever heard about Superfolded GFP? It is 50% brighter than GFP! And mScarlet is almost 6 times brighter than mRFP! How do I know? I look at this fantastic database called FPBase.
You can see which fluorescent protein is monomeric, sort them by excitation and emission or find which bleaches least or maturates fastest! Great tool! 🙂
Fluorophores are constantly being developed. If you make a new plasmid, make sure you check that the one your lab has been using for trillions of years is the very best one!
If you want to image a large piece of tissue, it is sometimes difficult to get it to stay still in a dish while you are imaging it, especially if you have medium on top.
One nice way is to make a silicon well around it, fill the well with medium then add a coverslip on top. This allow you to keep your sample for a long time.
Twinsil by Picodent works nicely. 🙂
Yet another chance to try RNA labelling: The FENO facility, here in Flemingsberg, has purchased a machine to multiplex RNA scope. They will present it on the 16th of October. 🙂
Here is the announcement.
We got tipped by one of our users of a robust way to label and image RNAs in cells and tissue. Here is the paper and one can buy the kit from here. Apparently it works a charm! Let us know if it works for you or not. 🙂
Someone telling you where to start your clearing experiments! Sounds like a dream paper, doesn’t it? 😉
Here is the link.