How to perform-control immunostaining experiment - microscopist subjective point of view. Pawel Pasierbek

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1 How to perform-control immunostaining experiment - microscopist subjective point of view. Pawel Pasierbek Immunolabeling and fluorescent detection became such a standard procedure in the biomedical research that a lot of us don t think about how artifact-prone it might be. I decided to make a very short subjective guide on how I would perform the immunolabeling experiment involving fluorescent detection. Please, don t consider it as a protocol, rather as a material to think about. 1. Find-out which microscope is best for you. Before you do any wet lab work determine which optical system will be best suited for your scientific question. There exists a big variety of microscopes which one can use each of them has its strengths and weaknesses. Talk to the staff of the BioOptics Core Facility to find out the best instrument for your experiment. The choice of the instrument might determine some technical details concerning the labelling/probe mounting procedure. 2. Which fluorophores can I use for the detection? In order to answer this question you need a negative control a sample that you processed the same way as the real experiment, but without using primary and secondary antibodies (AB). This allows you to check if your sample shows any autofluorescence and if yes, which fluorophore should be avoided for detection. Remember, the longer the wavelength of the fluorophore the worse the obtained resolution. Also establish which dyes can be excited best by the available lasers.

2 Fig.1. - Image of the negative control A,B,C,D DAPI, Alexa 488, Alexa 568, Alexa 633 channel respectively. Note weak signal in the DAPI channel it turned out that the blocking solution was contaminated with DAPI fluorophore. 3. Did my staining work? If you are detecting a protein of unknown localization, you have to be able to prove that what you see is real. In such a case you will need another control sample, namely one that includes the secondary AB only. Obviously you expect to see no signal at all since there is no autofluorescence (see above) and you washed the sample thoroughly. If you get a signal (and you are sure that there is no autofluorescence) this means that your secondary AB binds unspecifically to your sample. Fig.2. - Image of immunostaining with secondary Abs only. A,B,C,D DAPI, Alexa 488, Alexa 568, Alexa 633 channel respectively. Note the unspecific binding of Alexa 568 and Alexa 633. DAPI contamination present. In this case you should follow a rule: Solution to Pollution is Dilution Titrate the secondary AB don t follow the recommended dilution be opened and try 1:1000, 1:2000, 1:5000 etc. Wash the slides/sample in a big volume of washing solution with a detergent. I usually put slides into a metal rack and was them in a 500ml beaker gently agitating. Change the washing solution many times (10x 10min each does sometimes a trick), place only few slides widely spaced in the rack.

3 If you do immunostaining of samples placed/grown directly on the coverslip (which is the proper way of making a slide for microscopical experiment) use a reverse action tweezers and a wash-n- Dry Coverslip Rack Fig.3. Image of useful tools for immunostaining experiments: washing rack for slides, coverslips, reverse action tweezers. Never wash negative controls with the experimental samples together!!! Try different blocking agents. Sometimes it helps to try another batch /lot of the AB. Making a fresh dilution is also a good idea.

4 Fig.4. Left Image of immunostaining with standard diluted AB, 3x washed (PBST, 5min). Right Image of immunostaining with the same AB, properly titrated and washed. Notice improvement of contrast resulting in details appearing. 4. What about multicolor labelling? In case of multicolor labelling do single staining controls this will allow you to exclude bleed trough of the signal from one channel to another. Choose the fluorophores which are spectrally well separated. In case that you have to use ones which spectra overlap try spectral unmixing (here we can help a lot). It is preferable to combine dyes that can be acquired with one beam splitter (i.e. GFP + mcherry can, YFP + mcherry cannot). Often this means using the standard blue/green/red/far-red dyes. When spatial relationships are of interest, the most important targets should be labeled green and red, using blue and far-red only if additional colors are necessary, because the chromatic aberration increases sharply in this order. 5. Few further tips and tricks Before applying the AB to the sample spin-down the working solution of the AB for 30sec-1min at full speed and take the few µl from the top. Never vortex AB solutions! To save AB (primary as well as secondary), you can perform the staining covering the sample with a plastic coverslip, cut-out of the autoclavable bags it is for free, very flexible and can be cut to the desired size. Obviously you place the slides in a moisture chamber to prevent evaporation of the staining solution and do the incubation either at room temperature (RT) or overnight (ON) at 4 o C. (image of a coverslip) Once the immunostaining is done, you can counterstain the DNA usually done with DAPI. It is better to stain for DAPI separately. Embedding media that contain DAPI tend to give autofluorescence. Do a post fixation (e.g. 4%PFA, 4min for cell monolayers) to crosslink the AB (don t do it if you acquire a signal from a fluorescent protein only, since fixatives reduce the signal strength is such a case). Before putting the embedding medium, do a final wash with a distilled water to remove salt that can precipitate. Avoid using curing embedding media since they can change the 3D architecture of your sample. If you use the non-curing ones, apply nail polish or fixogum (rubber cement) to seal the edges of the coverslip. Fig.5. Fixogum for sealing coverslips.

5 It is worth to compare the results of the immunostaining with the localization of the fluorescently tagged protein of interest (hoping that tagging doesn t change the protein distribution in-vivo), in order to be sure that the AB does the right job. Once again remember to include controls in your immunostaining experiments. Yes, controls can ruin your experiment but they can save you a lot of time as well. Careful analysis of results of controls and experimental data is the only way you can draw conclusions from imaging experiments. 6. Where to buy tools for immunostaining experiments Wash-N-Dry coverslip rack e.g. Sigma Z EA Wash-N-Dry slide rack e.g. Sigma Z EA self-snapping forceps e.g. reverse action tweezers, ideal-tek I-2AX.SA (Compumet AG, Theodorshofweg 22, CH-4310 Rheinfelden) Fixogum can be found in the institute general store