Do you know that clearing is not just about light sheet microscopy? Even if you have done your job well and your sample is directly on the coverslip (not on the slide), as soon as your sample is thicker than 10 um (1 cell diameter), you will see the effect of the refraction index mismatch.
What is that? Your sample and the mounting medium around it have a certain refraction index (or likely several). The objective you are using is designed for a certain refraction index (e.g. air, water or oil). If these refraction indices do not match what happens? as soon as you image a tiny bit away from the coverslip, the sample will look elongated, the intensity and contrast will drop very fast.
Sounds familiar? If yes, changing your mounting medium to match the objective will solve the problem. It works for light sheet but it also works for wide field or confocal imaging! Just change your mounting medium and you will see an enormous difference!
Here is an article describing a one-step clearing protocol. This basically is about using a different mounting medium. Easy, cheap and non-toxic! Give it a try!
Have a look at this post for more info.
Ever wondered if that antibody you used throughout your whole PhD was actually also binding to something else than its supposed target protein?
Antibody validation in tissue staining is a very difficult task!
Here is a great step-by-step validation protocol published by EuroMabNet, a network of scientists who try to improve antibody validation.
And this paper gives a useful flow chart for antibody validation.
And here is the 5 pillars of antibody validation paper which explains what can be done to validate antibodies.
Here you can see a nice film of a beating cardiomyocyte.
It was transfected using Fuse-it vesicles full of the mRNA of LifeAct-tagGFP2. According to Ibidi, it also work well with primary cells which are typically difficult to transfect.
RNA-based transfection seems to be gaining speed compared to classical transfections using DNA.
If you try it, leave some comments here to tell us how it worked for you! 🙂
Sounds good! No more buffer that stops working after one hour of imaging!
This company sells SaraFluor650 secondary antibodies. This fluorophore seems to be a natural blinker which does not need to reducing environment. Apparently it doesn’t need very bright lasers either!
Apparently they have also developed a green variant. So you can run 2-colour direct STORM on your favorite TIRF system! 😀
They also sell pH sensors and more so check their website and if you try, write a comment to let us know how things worked!
It is not easy to find enough antibodies that work together to be able to label a sample with more than 4 antibodies at the same time. And even 4 is pushing it.
This paper describes a new immunostaining multiplexing method called 4i. The method is based on a special imaging buffer that prevents the antibody from being strongly bound to the sample due to the imaging process. This allows the authors to detach the antibody with gentle treatments, leaving the sample in a good shape and ready for another round of labeling and imaging.
Using this method they have successfully labelled the same sample with 40 different primary/secondary of the shelf antibody pairs!
The LCI will host a seminar about GlyCLICK, a new way to directly label primary antibodies and stop using secondaries: 6th of November at 13:00 in the Lipid seminar room in Neo, KI Flemingsberg. The seminar will be broadcasted live. Here is how to find us and here how to follow the seminar online.
Everyone agrees that it would be great to be able to label our samples without using secondary antibodies.
- less animals killed
- shorter and cheaper protocols
- no problem with isotype cross-reaction
- no problem with secondary species when using many antibodies at once
There are many kits to label primary antibodies directly with fluorophores. The main disadvantage compared to primary/secondary stainings is that direct labels are often weaker because the final primary/fluorophore ratio is too low. Using an amplification method like TSA (Tyramide Signal Amplification) leads to a loss of resolution.
Over the years users at the LCI have tried this kit, this kit and this one. They gave mixed results depending on the antibody but we keep looking! Come to the LCI seminar about a new direct labelling technique that uses Click chemistry.
Zell Kontakt is a German company making fantastic glass bottom dishes and plate. Check out why the LCI facility likes their products (absolutely no commercial interests!).
Great products but crap communication skills! Either their website is down or they do not answer the phone and emails and anyway if they do, make sure you have a German colleague around because no one speaks English.
Now we have found a way around the problem: MoBiTec sells Zell Kontakt products. 🙂
If you are at KI and need to find a KI-certified supplier, just come and ask us. 🙂
Measuring the volume of a cell is often done by labelling the cell membrane or its cytoplasm. Analysing large flat cells this way is easy but it is much harder for tiny cells like blood cells, yeast or bacteria.
Another way to measure volumes is to use a negative stain, i.e. where the medium is made fluorescent with a dye that does not go into the cell. The cell appears as a black hole in fluorescent images and unlike lipid-based membrane labelling, borders are even and easy to segment.
While many dyes can be used for live cells, one must choose large dyes when negatively imaging cells that have been fixed and permeabilized.
This paper and this one use high molecular weight (2000 KDa) Dextran to achieve these results and measure the size of bacteria.
This recent paper optimizes the technique.
Here are a few image analysis tools (constructs for FRET, software…) that might be useful to you.
- Construct for fluorescence biosensors and optogenetic tools
- Free Image analysis software
- Free Image analysis software
- Free Image analysis software
Tomorrow (17 sept) we will enjoy a seminar and a live demo about the Crest V3 spinning disk confocal which is being set up at our facility as I write! 😀
Very cool confocal!
- enormous field of view (32 mm diameter)
- fully confocal
- can image at 100 frames per sec
- spits out Nyquist resolution with the 60x objective!
You can come to the seminar (at 10 in the Gene seminar room at the LCI facility) or listen to it remotely (see here how to follow the LCI webinars).
You can even book a private demo to image your own samples.