One antibody is used in our lab so often that we each keep our own bulk stock! This antibody is S6 Ribosomal Protein (5G10) Rabbit mAb #2217, which derives its utility from 5G10’s unique antigen. This blog will discuss how we use 5G10 as a readout for sample quality as well as an indicator of a well-executed IF experiment. In this way, 5G10 helps us produce the best quality data and images possible. It is our hope that you will be able to use these tips in your lab to increase confidence in your results.
The unique composition of 5G10’s antigen, which is rich in aldehyde-sensitive lysine residues, makes the binding of this antibody sensitive to formaldehyde fixation. This has been characterized empirically through in-house testing in formaldehyde fixed cells, though is not the case with formalin fixed paraffin embedded samples (possibly because of antigen retrieval often done with this sample type). The results of these tests demonstrate that 5G10 only works when samples have been fixed between 5 and 30 minutes with 4% formaldehyde. As shown in the image panel below, under-fixation or over-fixation will not yield sufficient signal. The inability of 5G10 to stain S6 is an indicator that the sample may have been under or over-fixed. Improper sample fixation can lead to suboptimal results with other antibodies as well.
This is particularly useful when staining tissue. The varying rates of fixation for different organs mean they may each achieve a different state of fixation if all fixed in the same batch. This is referred to as the ‘penetration-fixation-paradox’ in the literature (1). Typically we utilize perfusion fixation when working with rodent models. This approach, though much faster than immersion fixing, can also yield variability in formaldehyde penetration when done improperly. Positive 5G10 staining throughout a tissue gives us confidence that our sample has been properly fixed. The image below depicts inconsistent S6 signal in sections taken from two different livers. The weak and spotty S6 signal in the image on the right suggests that the tissue is not within the desired range of fixation and should not be used for IF staining. The image on the left demonstrates the expected S6 signal in properly fixed liver, demonstrating the tissue is of satisfactory quality for use.
5G10 has another trick up its sleeve! Low-intensity signal with this antibody may not always be a result of poor fixation but instead improper buffer pH. Buffer preparation is an often underappreciated element that can make or break an IF experiment. In the experiment below, we demonstrate how decreasing buffer pH can negatively affect the signal intensity with 5G10. Combined with its sensitivity to fixation, this antibody is a great all-around control for the staining process.
In this blog post we have discussed the utility of S6 Ribosomal Protein (5G10) Rabbit mAb #2217 as a means to validate the efficacy of your IF staining as well as an indicator of properly fixed tissue. Here in the IF group at CST, we are committed to producing data of the highest quality; using 5G10 as a control in all of our immunofluorescence experiments is a step we take to help identify issues with sample fixation or the staining protocol. Hopefully you can try using 5G10 as a control in your future experiments as well!
(1) Thavarajah, Rooban et al. “Chemical and physical basics of routine formaldehyde fixation.” Journal of oral and maxillofacial pathology : JOMFP vol. 16,3 (2012): 400-5. doi:10.4103/0973-029X.102496