Seminar on how to directly label your primary antibodies

Skip the secondary, part 3! 😀

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.

Supplies of Zell Kontakt products

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. 🙂

How to precisely measure the volume of a cell?

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.

 

Free genetic and image analysis tools

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

http://www.hahnlab.com/tools/index.html

  • Free Image analysis software

https://www.utsouthwestern.edu/labs/danuser/software/

  • Free Image analysis software

http://cismm.web.unc.edu/

Crest V3 spinning disk confocal demo

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.

Matching the refraction index of live samples

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.

How to identify cells and nuclei in an image?

NucleAlzer is a great new deep learning tool to identify roundish objects like nuclei and cells in fluorescent or bright field images.

To test if the tool works for you before you download it, you can simply upload one of your images and check the result. Easy! 😀

Super-Resolution spinning disk demo at the LCI!

Dear microscope freaks

How would you like to run some gentle live sample imaging with a 60x objective with:

  • an xy resolution of 120 nm without software tricks (or even better after deconvolution),
  • the great contrast of a true confocal,
  • 82 frames per second,
  • or decide to bypass everything, go widefield and image at 100 frames per second with a super large field of view (220×220 um)?

Sounds good to me! 🙂

For the next 2 weeks you can do that with the new toy on demo at the LCI facility!

The beast is a new sort of spinning disk confocal and is called SoRa (Super-Resolution Optical Reassignment). It is a collaboration between Nikon and Yokogawa.

We even have 2 cameras to compare (Prime95B and BSI from Photometrics).

Oliver Garner from Bergman Labora will give a short online presentation of how SoRa works on Monday (29th) at 13:00. The presentation is done remotely and broadcasted live. You can join the audience from the comfort of your office chair by following the instructions here (please try beforehand to make sure all works).

Interested in trying it? Please contact us.

A dream!!

LCI product seminar: How to label organelles in live cells?

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! 🙂

CUBIC seminar tomorrow at Scilife

Do not miss the seminar tomorrow morning at Scilife: the inventor of the CUBIC technique, Etsuo Susaki, will present his technique which can clear fatty and dense tissues.

Click here to know where and when.

And here is the link to the updated Cubic protocol.