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.
Have you ever heard about Superfolded GFP? It is 50% brighter than GFP! And mScarlet is almost 6 times brighter than mRFP! How do I know? I look at this fantastic database called FPBase.
You can see which fluorescent protein is monomeric, sort them by excitation and emission or find which bleaches least or maturates fastest! Great tool! 🙂
Fluorophores are constantly being developed. If you make a new plasmid, make sure you check that the one your lab has been using for trillions of years is the very best one!
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. 🙂
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. 🙂
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! 🙂
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!