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. 🙂
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:
- Gelatin-coating solution: 1 L deionized H2O, 5 g gelatin, 0.5 g chromium potassium sulfate dodecahydrate CrK(SO4)2 · 12H2O
- Filter units
- Histological slides
- Hot plate with magnetic stirrer
- Slide racks
- Staining dish
- 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.
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!! 🙂
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.
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. 🙂
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!
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! 🙂
Next wednesday morning, David Unnersjö-Jess from KTH/Scilife will give a talk at the Live cell imaging facility (9:30, DNA seminar room), about a new and exciting aspect of microscopy: Expansion microscopy.
This is a technique where one ‘blows up’ the sample while keeping all proteins in place and at the same relative distance from each other. The sample is simply ‘inflated’. One can then take images of it with a normal microscope but the resulting image give a much higher resolution than normal microscopy.
After the seminar, David will have an open discussion with anyone who would like to try the technique.
On Thursday (15th of March), Teng-Leong Chew, director of the Advanced Imaging Center at the Janelia Research Center (Virginia, USA), will come to Stockholm and present what his facility can potentially do for you.
The AIC offers a crazy service where visiting scientists can use the super resolution systems they develop there with the help of their experts. This service is free of charge, including accommodation.
This can allow you to quickly run a project involving STED, PALM/STORM, SIM, adaptive optics or super resolution lattice light sheet microscopy.
Together with Leong’s presentation of his facility, there will be a few seminars by the Live Cell Imaging facility as well as several parts of the Advanced Light Microscopy facility at Scilife.
This is a great opportunity to catch up with what is available to you here and in the US. Hope to see you there! 🙂
The next big thing in microscopy comes straight from the sky! Apparently astronomers have been using adaptive optics for years to improve their images and it is only getting into our microscopes now!!
Adaptive optics takes the nightmarish situation seen in b and puts it back straight as in a! This is done by measuring the wave front and deforming a mirror to reshape the wave front to perfection!
Sounds like a dream but I have actually seen it in action at the 2016 AQLM course and there was a definite WOW effect! 🙂
Check this review to learn more