The Live Cell Imaging facility
@ Karolinska Institutet, Sweden
Some of our users have tested the ProteinTech FlexAble kit to directly label primary antibody and they are very happy so we thought we should share! 🙂
It only takes 10min to label the primary. It saves time for the sample staining procedure and it saves cute animals when one stops ordering secondary antibodies! 🙂 In the hand of our users, the images were as bright or brighter than the same labelling with primary + secondary.
And you can ask for a free sample to test! :):):)
Two really nice symposia/workshop on clearing and expansion microscopy next week!
Fluorophores are in constant development.
There is now a new generation of Alexa Fluor fluorophores: the Alexa Fluor Plus. They can be purchased already coupled to secondary antibodies on the ThermoFisher website.
Brilliant violet antibodies are also very bright and despite their name, they come in all colours. The can be purchased from Biolegend which produces the fluorophore.
Another excellent source of bright fluorophores is the Janelia Fluor dyes. They can be purchased coupled to antibodies from the NovusBio website.
Hans Blom from the Advanced Light Microscopy facility at Scilife organises next week (10th and 1tth of June) 3 sessions of great seminars:
I especially recommend the CUBIC seminar to anyone who wants to image samples thicker than 100 um. This will teach you how to treat your sample to make it fully transparent so you can image through many cm of tissue!
🙂
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.
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!
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.
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.
To image a thick sample, it is crucial to match the refraction index of the sample with that of the immersion medium between the sample and the objective. Typically, life samples are in an aqueous solution like culture medium which has a refraction index of 1.33. Unfortunately organoids often have a higher refraction index closer to 1.44 therefore as one images deeper into the organoids, light scatters due to the refraction index mismatch and the images become blurry.
This paper presents a product that has a high RI and is compatible with cell culture. Good to keep in mind for those who image organoids over time.
On Wednesday (13th) at 9:30 in Lipid seminar room in Neo (KI Flemingsberg), please come and enjoy a short seminar presenting a new way to label organelles in live cells.
LabLife will present their product called Viromer Cytostain.
We will stream the seminar live so you can follow it even from your desk! 🙂
