Immunofluorescence pointers

1. Antibody specificity - Please ensure that you’ve done adequate controls to confirm that your primary and secondary antibodies are specific for your antigen.
   This includes doing

   1. a knockout or knockdown control to ensure that your primary is actually recognizing your protein of interest.
   2. a no-primary control for your secondary to see how it stains your cells.
   3. a co-stain where you omit each primary antibody while including all secondaries to ensure that your secondaries are not reacting with the incorrect primary antibody. Purchasing highly cross-subtracted antibodies such as the donkey secondary antibodies made by Jackson immunoresearch is usually essential when trying to use primary antibodies from closely related species (i.e. rat and mouse).
2. Antibody dilutions - When you are setting up staining protocols, it is useful to determine the lowest dilution of primary and secondary antibodies that produce a real signal. Using too much primary and secondary antibodies can lead to aberrant staining. In addition, incorparate DAPI into your secondary antibody staining. Don't use a mounting media with DAPI, because mounting media with DAPI creates more background.
3. Fixation - There are many different fixatives that can be used for immunofluorescence. Paraformaldehyde (PFA), methanol (MeOH), glutaraldehyde, acetone, and glyoxal have all been used successfully. It is frequently beneficial to test several different fixatives to find out which one best presevers your structure of interest as well as the antigenicity of the protein that you are looking for. It is also possible to combine fixatives to enhance their efficacy. For instance, a mixture of 90% methanol with 10 % (by volume; 3.2 % concentration if working from a 32 % stock) PFA works very well for fixing the cytoskeleton. Note that PFA and glutaraldehyde work the best at a basic pH so using a basic buffer like PHEM at pH 8.0 or adding some sodium bicarbonate to MeOH + PFA will enhance the fixation. After fixation it is usually best to get rid of unreacted aldehydes by washing with a buffer with a primary amine like Tris or by incubating with (freshly-prepared) sodium borohydride. Fixative quality also impacts how well it works. Freshly prepared PFA or glutaraldehyde diluted from a freshly opened concentrated stock will produce better results.
4. Mounting - Although mounting media is usually the last thing that scientists consider when performing immunofluorescence, the selection of the proper mounting medium is essential for getting the most out of the samples that you work so hard to optimize. Ideally, you want a medium that will preserve staining and combat photobleaching during imaging.
   1. Don't use a mounting media with DAPI, because mounting media with DAPI creates more background. Instead, incorparate DAPI into your secondary antibody staining.
   2. You should consider the refractive index (RI) of the medium. If the RI is too high (such that it matches or is close to the glass of the coverslip; RI = 1.5), the Perfect Focus System (PFS) will not work properly since the PFS is looking for the change in RI going from the coverslip to the medium.
   3. The ability of the medium to maintain staining is important and should be tested. We have observed that some types of staining are very labile in one type of medium but permanent in another.
   4. Some media will harden over time, but others won’t. If you select a hardening medium, you should evaluate whether this causes the sample to flatten and/or contract.
   5. If you seal your coverslips with nail polish, please ensure that the nail polish is fully dried before you start imaging. CoverGrip™ by Biotium is superior in many ways to nail polish. Nail polish can contain chemicals that can leach into the mounting medium and either fluoresce or diminish your sample fluorescence.
   6. After your coverslip is mounted and stable, you should wash its surface with distilled water to avoid contaminating the immersion oil with salts.