Cool technique – Page 4 – The Live Cell Imaging facility

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

On Wednesday (13^th) 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.

Antibody-based technologies at SciLife

Apply to this course at SciLife and learn about Antibody-based technologies on the 25th of March.

Glyoxal instead of PFA

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. 🙂

How to stick a tissue section to a coverslip

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 (and pasted below).

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, either the thick glass or the mounting medium end up between the objective and the sample. The objective is not designed for this. ?

Here is the protocol:

Reagents Required
Gelatin-coating solution: 1 L deionized H2O, 5 g gelatin, 0.5 g chromium potassium sulfate dodecahydrate CrK(SO4)2 · 12H2O

Materials
Filter units
Histological slides
Hot plate with magnetic stirrer
Slide racks
Staining dish
Thermometer

Procedure

Prepare the gelatin-coating solution by dissolving 5 g of gelatin in 1 L of heated, deionized H₂O (temperature should not exceed 45 °C).
After the gelatin has dissolved, add 0.5 g of chromium potassium sulfate dodecahydrate. Chromium potassium sulfate dodecahydrate will positively charge the slides allowing them to attract negatively charged tissue sections.
Filter this solution and store at 2-8 °C until use. It is recommended that this solution be filtered again immediately before use (adjust to room temperature before filtration).
Place the histological slides into metal racks.
Note: The slides should be cleaned by washing them in soapy water and rinsing them thoroughly, first in tap water and finally in deionized water.
Dip the racks containing the slides 3 to 5 times (~5 seconds each) into the gelatin-coating solution.
Remove the racks containing the slides and let them drain. Blot excess solution from the racks onto filter paper (gently tap the racks against the filter paper for better drainage).
Place the racks containing the slides on the lab bench and cover them with paper towels to protect them from dust.
Dry at room temperature for 48 hours.
Dried slides can be put back into the boxes that they arrived in and stored at room temperature until use. Slides intended for cryostat sections can be stored at -20 °C.

Great video tutorials for our Primo users and paper on microniches

Hello again

Here you can see very nice video tutorials on the Alveole website and this is a cool article by Viasnoff et al about making 3D microniches with 1 um resolution! And you can do this at the LCI facility!! 🙂

More about RNA labeling

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.

Robust RNA labeling in cells and tissue

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. 🙂

Clearing and expansion microscopy course at Scilife in September!

Expansion microscopy and clearing are fantastic tools for anyone who images thick (> 1 cell diameter) fixed samples. We are now lucky to have access to state of the art talks and hands-on workshops at Scilife in September. Register early not to be disappointed!

Please apply to Hans (first day) and David (second day) directly!

Skip the secondary, Part 2! :)

In one of the LCI earlier posts, you can read about easy and powerful it is to skip using a secondary antibody while still having a bright signal.

We have not had any feedback on the Kromnigon technology but we got to hear great praise and see superb images of a similar product called Mix-n-Stain by Biotium.

You can now directly label your stock of primary or favorite tag ligand (Snap, Clip, Halo, TMP) in just 30 min! Each primary gets 3-5 fluorophores according to the Mix-n-Stain brochure so there is no problem with dim directly labelled antibodies as used to be the case in older labeling technologies.

Skipping the secondary means gaining time but also no more headache about matching antibody and tissue species so you can stain your tissue with 7 or 8 antibodies if you image with narrow filters or spectral unmixing.

Bye bye ‘No primary’ controls! It is high time to switch to an isotype control: buy an antibody with the same isotype as your favorite antibody and label it in the same way. This allows you to detect any aspecific binding of your primary antibody.

Last but not least, skipping the secondary means less animals used to produce them. That alone is a bit plus!

It costs 100€ or so to label 50 ug of antibody. Definitely worth a try! 🙂