Background information

Although knowing your microscope and the physics behind obtaining the best quality image is important, excellent sample preparation and labeling is crucial because without it, you will not be able to obtain the data about the biology, no matter how good your microscope is. 

 

There are 2 types of samples: 

(1) live cell:- which relies on fluorescent proteins or vital dyes which need to be titrated accordingly to ensure it does not interfere with the cell biology. 

 

(2)  fixed cell / tissue:- which can incorporate fluorescent proteins, or tags such as SNAP-tag, HALO-tag and CLIP-tag, or antibody based labeling 

 

Labeling your fixed sample with fluorescent antibodies (immunohistochemistry: IHC, immunofluorescence: IF) is both an art and a science as there are considerations at each step:  

  1. Choosing the correct antibody clone 

  2. Ensuring there is the correct positive and negative staining controls, which can also require finding alternate samples. 

  3. Choosing the optimal multi-colour fluorescent combination 

  4. Using the correct fixative for the right amount of time on the cells or tissue to preserve the tissue structure 

  5. Using the right antigen retrieval method to preserve the epitope 

  6. The adequate permeabilization method to allow the antibody to penetrate 

  7. Quenching endogenous substrates that could interfere with your antibody binding 

  8. Labeling the sample with the antibody with the optimal concentration and staining buffer 

  9. Mounting the sample in the right media to not distort the structure and preserve the fluorescence ready for image analysis. 

 

Most new-comers to sample preparation do not recognize the amount of optimization required to produce a good fluorescent sample, but there are many resources to help. 

 

IHC World: provides protocols and information for beginners to experts 

Abcam: provides high quality antibodies rigorously tested for IHC 

Nature Protocols for Confocal microscopy. 

 

We have also provided a basic protocol for frozen sections that includes tyramide signal amplification (TSA) steps  that can help amplify the fluorescent signal around a lowly expressed protein of interest.