Dear Eleanna,

1. Tween 20 is a detergent, a strong one though. It is needed in the washing steps to remove non-specific binding of the antibody. This is very crucial in any fluorescence staining experiment since flourochrome binding is highly sensitive, so tends to give background much higher than other methods. However, I would recommend using Tween-80 since it is a milder detergent.

2. Permeabilization of membrane is a critical process and if done in an uncontrolled manner, may lead to cell damage as well. Therefore, it is better to do at 4 degrees. The time, however, I think 5 min would be little too less at 4 degrees. We do it for 15-20 min at 4 degrees or 5 min at Room temperature.

I hope I tried to answer both your queries. All the best.

Dear Eleanna,

1. Tween 20 is a detergent, a strong one though. It is needed in the washing steps to remove non-specific binding of the antibody. This is very crucial in any fluorescence staining experiment since flourochrome binding is highly sensitive, so tends to give background much higher than other methods. However, I would recommend using Tween-80 since it is a milder detergent.

2. Permeabilization of membrane is a critical process and if done in an uncontrolled manner, may lead to cell damage as well. Therefore, it is better to do at 4 degrees. The time, however, I think 5 min would be little too less at 4 degrees. We do it for 15-20 min at 4 degrees or 5 min at Room temperature.

I hope I tried to answer both your queries. All the best.

Hi,

G. Anupa answered your questions great, but I would add few information:

Everything in immunocytochemistry depends on type of your cells and desired proteins which you want to detect. I tried to perform immunocytochemistry with formaldehyde/paraformaldehyde/Tween 20/ Tween 80 for almost a year with different combinations, incubation times, concentrations and this simply did not work well.

Then I tried ice-cold acetone and it works great, 5 minutes in 4 degrees and cells are fixed and perneabilized. (but you have to check the time properly, never use more than 5 minutes using acetone). So how long you perneabilize depends on the method you use and proteins you would like to detect.

For blocking is great bovine serum albumin with glycine in phosphate buffered solution.

I got first beautyfull picture from immunocytochemistry after 18 months of trying (it was only my hobby method) :)

good luck

Depending on your sample, you may want to try different detergents. For cells (monolayer on a coverslip), I typically use .1% Saponin (not as harsh of a detergent) at RT for 1hr to achieve permeablization. For brain slices, I typically use .2% TritonX 100 (stronger than Saponin). These can also be used in your washes to remove non-specific staining as mentioned above.

Yes I totally agree with Matej Vicen and Elliot. It much depends on the cells you are handling.

Dear Eleanna,

I can just agree with the above mentioned. For different purposes as different tissue, cells or different staining/ imaging methods I use different protocols.

To your first question:

During my PhD thesis I used 0.1% Triton for 10 min at room temperature and PBS containing 0.1% Tween for all washing on spinal cord slice.

Now I work beside in vivo imaging with kidney cell lines for super resolution microscopy (STED and STORM). For this I adapted a completely different protocol in which I stopped working with Tween at all. I still permeabilize with Triton 0.1% because I found Saponin not effective when you need to stain innermitochondrial proteins.

If you use PFA/ Formalin or acetone depends if your epitope is prone to cross linking since formalin cross links Lysine residues while acetone is basically drying out the cells. The former has the big advantage of better morphological preservation since you don't get that much shrinking artifacts but it might interfere with antibody binding by cross linking residues in your epitope of choice.

To your 2nd question:

Every chemical reaction depends on temperature and often the warmer the faster the reaction. You would wonder what 5°C can make a difference. The term room temperature is not really precise and the room temperature in your lab might change due to seasonal temperature changes quite a bit. So in one lab room temperature might be 28°C due to direct sun light heating up the room in summer with no air conditioning in place and in others it is 18°C.

With 4°C in the fridge you have a standard temperature if your fridge is stable at that temperature which makes your protocol more reproducible. I would say you get an even harsher permeabilization at room temperature with Triton which has an influence on the cell membrane. If you want to look at membrane proteins keep it brief. If you are looking for cytosolic or organelle proteins a bit longer is not a problem I would say.

I know from my PhD time a lab in Glasgow doing an amazing job with immune staining in the spinal cord. If you need advice maybe they could help. It is the group of Andrew Todd. https://www.gla.ac.uk/researchinstitutes/neurosciencepsychology/staff/andrewtodd/

Cheers,

Claus

Thank you very much everyone, this helps a lot.

Here's a good page that I think is helpful: https://www.labome.com/method/Detergents-Triton-X-100-Tween-20-and-More.html

First and foremost purpose of using a detergent, such as Tween 20 or NP 40 etc.,) is to prevent non-specific binding of antibodies (primary and secondary) to tissue or cell pellet, thereby avoidance false positively.