How can you improve your antibody staining protocol?

Carefully choose epitopes of the antibody against when validating gene knock, especially when there is residual protein expression.

Make sure to check the host in which the antibody is grown. If your sample comes from the same organism as the host of your primary of choice, reconsider going for primary harvested from another host. If not, make sure you used F(ab) after blocking to prevent non-specific binding.

It is important to know that some epitopes will change post fixing so much so that it is no longer recognized by the antibody. In this case one could try different clones of the antibody or two different fixatives to get around this issue.

For anything cytoskeletal, PFA or formaldehyde works good, anything other than cytoskeleton, you can use methanol for staining. However keep in mind methanol dehydrates the cell so if this is some embryo (or single cells or organelles) that you’re staining, you need to be extra careful while changing the antibody to washes. There are a lot of antibodies that stain better with methanol fixative.

Wash your samples with a low concentration of detergent, like 1% Tween or 0.1% Triton diluted in PBS or TBS to aid with permeabilization

Each and every step in antibody staining has a principle and it's important to understand the logic behind these steps. For instance, using the right blocking buffer: BSA is one of the most commonly used blocking agent but the primary antibody I was using was generated using an antigen where the molecule was conjugated to BSA. So, in this case, using BSA as a blocking agent will give rise to a huge background noise. So, I chose normal goat serum (NGS) as my blocking agent. It is absolutely important to know your chemicals and their compositions before doing an antibody staining experiment!

It's important to know the "how and why" for each step and then think of inverting this to understand why something is not working. For instance permeabilization is a key step to enhance accessibility of the target, but ask yourself " how are the other ways my target can be masked?"- suddenly protein complexes, organellar sequestration become important factors to consider.

When testing a new antibody for immunocytochemistry/immunofluorescence, consider testing it at different concentrations before deciding on the best protocol for your sample. Sometimes a lower concentration of antibody can give better results than a higher one.

If you’re staining smaller organelles such as a centrosome or some granules etc, reusing the primary antibody the second time helps a lot to get rid of any background. Primary antibody once made and stored in the 4C can be re used up-to 3 times (3 experimental replicates) within 14 days of making it.

Some ways to minimize non specific binding would be to wash between the primary and secondary antibody. Make sure you have a good 10 mins x3 washes with the blocking buffer between your primary and secondary antibody. If there is a dye or stain that has chances of bleed through such as dapi, I prefer to add that after the secondary antibody (instead of with it).

Use 1-5% w/v serum from the source species of the secondary antibody as a blocking buffer component to prevent non-specific binding of the secondary antibodies

With confocal images, it helps to separate each channel during post-acquisition in an image analysis software to evaluate any non-specific or background signal. If a signal is detected uniformly across all channels in a multi-channel image from different laser modes, chances are they are non-specific. Spectral unmixing can help with separating autofluorescence or background signals

If you can - add a positive control - nothing worse than poor antibodies (not binding at all or very low sensitivity!). Also add a negative control - to cover non-specific signal, which may lead to rabit holes!