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.
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! 🙂
Together with the coverslip and the immersion medium (oil, water, glycerol or air), the sample mounting medium is part of the design of a microscopy objective. Matching the refraction index of the sample to the one recommended by the manufacturer of the objective will make the sample transparent for the objective, drastically improving fluorescence microscopy in samples thicker than a couple of um (i.e. anything except fluorescent beads!).
Not matching the refraction indices is equivalent to watching something through a wet window… Far from optimal! :-/
The refraction index recommended by the manufacturer is the same as the RI of the immersion medium: 1.52 for an oil immersion objective, 1.47 for a glycerol objective, 1.33 for a water objective, 1 for an air objective.
This article compares 7 mounting media and their effect on the refraction index of brain samples. CFM3 seems to be a cool mounting medium. The company that produces it has partially paid for the study but it sounds worth a try anyway!
In the same vein, this article presents a non-toxic way to change the refraction index of cell culture medium (not the sample) to improve imaging of live samples. Sounds pretty promising to grow live organoids which quickly become opaque. This will also be very useful when clearing samples as the sample chamber on a light sheet microscope is big so this is a cheap way to fill the chamber for imaging. 🙂
If you try any of these 2 chemicals, please leave a comment to let us know how it went! 🙂
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. 🙂
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. 🙂
Someone telling you where to start your clearing experiments! Sounds like a dream paper, doesn’t it? 😉
Here is the link.
Buying Histodenz powder from Sigma is rather expensive. Alternatively, you can buy Omnipaque from GE Healthcare. It is the same product but comes as a solution and costs around 2000 sek for 10 bottles of 350 mL!
One of the major pains with membrane dyes for live cells is that within 30min, all the vesicles inside the cells are also labelled. Another major pain is when you discover that your dye is not fixable…
The CellBright dyes label membrane is apparently fixable, even with methanol, and doesn’t do the flip flop thing! Potentially extremely useful for image analysis!
Please leave a comment to let us know how it went for you! 🙂
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! 🙂