We're experiencing very high background in the green (AlexaFluor 488) channel in immunofluorescent labeling of paraffin sections of rat brain. The high levels of autofluorescence are associated with white matter (presumably due to myelin). We have no problem in the blue, near red, or far red channels; and the problem appears only in sections of brain. Any suggestions that could help reduce this autofluorescence would be greatly appreciated. Technical details of the staining follow below.
Brains were fixed in 4% paraformaldehyde and subjected to standard paraffin embedding procedures. During the labeling procedure the sections are deparaffinized in Xylene, rinsed in ethanol, followed by sequential antigen retrieval in: ice cold methanol (20 min), 1% sodium borohydride (2 min, room temp), and Citrate buffer (pH 6; start at 95 degrees C and allow to stand at room temp in the buffer to cool for 30 min to 1 hour. After that we block nonspecific labeling with normal goat serum and then perform standard primary antibody and secondary antibody incubations and mount the labeled sections using Prolong Gold.
Autofluorescence is the bane of IHC. Many solutions are now available to deal with this.
See: http://tinyurl.com/ldrtxlb
You don't need to use expensive commercial kits for this. There are simple methods, using common, cheap rgts, such as Trypan Blue or Sudan Black.
See: http://www.ncbi.nlm.nih.gov/pubmed/21970489
Sodium borohydride (as you've used) often, but not always, works.
Also-- Bruce Pfeffer in my lab has used NON-ALDEHYDE fixation method, called "Methacarn".... it uses choloroform/methanol. See
http://www.nature.com/labinvest/journal/v80/n2/full/3780023a.html
http://www.cpl.colostate.edu/pathology/protocols/methacarn.htm
http://link.springer.com/article/10.1007%2FBF00306176#page-1
How about your antibody preparation. I highly suggest antibody diluent is used instead of normal buffer
Mohd, Very good point. Ineffective blocking can definitely lead to high background. I left that information out of the description in the original question above. We block in 10% normal goat serum + 5% BSA + 1% fish gelatin + 0.5% triton in HBSS for 1-2 hours at room temp. We use that same solution to dilute our antibodies. Its a very effective blocker.
I'll ask you something. We did IHC (Annexin V) for brain (dorsolateral preforntal coretx and cerebellum) and liver. As a result, we have same problem like David Sherry. If we wanna publish our IHC results, do we get a negative feedback and how we solved this problem?
I don't think you get a negative feedback. Many studies have similar results.
For anyone who may be following this question, we were able to pretty adequately address our autofluorescence problem using post-staining of the sections with Sudan Black after we finished the immunolabeling steps. Our experience is that this approach works best to reduce autofluorescence in specimens that are labeled for antigens that give reasonably strong immunolabeling. One must be careful not to overstain the sections with Sudan black, as this can mask some of the genuine immunolabeling or antigens that show only weak labeling.
The protocol we used is attached. The references that we adapted the procedure from are given at the end of the protocol.
You must be careful not to overstain the sections with Sudan black, as this can mask some of the genuine immunolabeling or antigens that show only bad mark labeling.
- Badges
- Science topic
- Similar topics
- Organizational Psychology
- Training