I bought two secondary antibodies, anti goat alexa fluor 488 (A11055), and anti rabbit Cy3 (A10520). I used them before with anti-neurofilament primary antibody and anti-GFAP primary antibody, and I didn't have problems. Now I am using them in spinal cord sections with two primary antibodies(goat anti Nogo A and rabbit anti Semaphorin) and I have a terrible background like many points. I attached an image. The primary antibodies incubation is 4oC for 18 hours. Could someone help me with this problem, thanks. I wrote the before but I didn't have answer. Thanks you, Lucía Botero
Dear Patrice, I have the problem only whit secondary antibodies. I did serial dilutions for the primary and secondary antibodies, I am using primary antibody at 1:50 and secondary antibodues at 1:500.
At what magnification is the image? As I understand, if the spots are cells, are they actually not supposed to express both Nogo A and Semaphorin?
The image is at 400x, some shot spots at the cellular membran could be Nogo A or Semaphorin but the most in this image are back ground, we can see the spots in both channels (green and red) in the same location.
Hi Lucia,
What species are these spinal cord sections from? I've seen a very similar autofluorescence called Lipofuscin in my older mice brain sections. It accumulates with age, so it is common in human tissues, and tissues acquired from older specimens. It is usually very punctate, bright, and can be seen across fluorescent channels.
A similar question was asked and answered on RG here:
https://www.researchgate.net/post/How_to_decrease_PFA-induced_background_fluroescence
I was able to deal with lipofuscin in my older mouse brain slices by using a 1-10 mM CuSO4 in 50 mM ammonium acetate buffer. It worked like a charm without affecting my fluorescent dyes. I would highly recommend it! It took super bright punctate background fluorescence, which obscured cell morphology and antibody staining, and left a bright signal.
The protocol can be found in the paper below (Schnell et al. 1999).
Best of luck!
http://www.ncbi.nlm.nih.gov/pubmed/10330448
Hi Leslie, thanks for your help, I have rat tissue sections( 6 months old). I already did another immunostainning with anti neurofilaments+alexa fluor 488 and anti GFAP+Cy3 with the same tissues and I didn't have problem. The unique change is that I stored the secondary antibodies at -20oC, and I don't know if it is a problem. Do you know something about this?
the washing of sections perfectly after incubation with primary and secondary antobodies is important 3x10 min with PBS or PBT. also may be you need to use a high dilution from the primary or secondary antobodies(try with different concentrations).
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