I am relatively new to immunofluorescence, and I am having trouble getting my assay working. I have a murine tumor that was formalin-fixed overnight and paraffin embedded. The tumor was sectioned at 5 um. My problem is my negative control is very bright. The image attached is of a negative control, no primary and no secondary antibodies.
Staining protocol:
1. Permeablize with 0.25% TritonX-100
2. Wash in PBS with 0.05% tween 20, 3 times, 5 minutes each.
3. Block with 10% donkey serum for 1 hour.
~ At this point, this section was coverslipped using Vectashield with DAPI, and imaged. I also have gone on to add primary and secondary antibody in other slides, but since I cannot tell the difference between the positive and negative controls, I did not include those pictures. Eventually I plan to use Alexa 594 and 488.
Veronica,
Many paraffin embedded specimens have a very high level of autofluorescence. I have attached a protocol for immunolabeling paraffin sections that works pretty well for my lab. It has a lot of technical detail in it that I've left out of the discussion below. We work on brain and retina mostly, but we've used this procedure for other tissues as well. Our experience is that autofluorescence is also wavelength-dependent with shorter wavelength channels being worse than longer wavelength channels (Blue>> green> red> deep red). You might be able to help yourself by avoiding the high background channels when possible.
There are several things that you don't mention, so I assume there are important things that you are probably not doing in your labeling procedure.
1. A critical first step to immunofluorescent labeling of paraffin sections is to get the paraffin out of the tissue. This will expose the antigen and allow your antibodies to penetrate better. Triton alone probably won't do the job on paraffin sections. This is typically done using xylene, followed by rinses in ethanol and then exchanging to water or other aqueous buffer depending on what the next step in your procedure is. Even after removing the wax, we still see VERY high autofluorescence levels, so we do an autofluorescence reduction treatment.
2. Autofluorescence reduction. We routinely treat our sections with 1% NaBH4, which breaks double bonds and helps reduce autofluorescence. There are other procedures out there, this is just one option. The protocol has the details and the recipe. An important note here is that NaBH4 should be dissolved in WATER not PBS, which will cause it to bubble violently and destroy your tissue. NaBH4 will generate hydrogen gas, so some care in handlinh is a good idea. If NaBH4 doesn't work very well for you, you should try another one.
3. Fixation and paraffin embedding often denatures antigens, rendering them hard to detect with your antibodies. You are likely to need an antigen retrieval step. Again there are a variety of procedures out there, and you may need to try several to find the one that is optimal for you. Our standard approach to antigen retrieval is to treat the deparaffinized sections in hot citrate buffer (pH 6, 95 degrees). The protocol has the details and the recipe.
4. Following antigen retrieval most procedures have a blocking step that will use normal serum or bovine serum albumin to block sites that antibodies might bind to no-specifically. These solutions typically have some detergent in them to help open up the tissue and expose the antigen. The blocking solution also can be used as the buffer to dilute your antibodies.
5. The primary antibodies are applied. You will need to work out the proper incubation length and antibody concentrations for your specific situation. Our standard is to incubate the sections in primary antibody overnight at room temperature, but that can be adjusted according to need.
6. Rinse the sections to remove unbound primary antibody and apply fluorescent secondary antibody. We use 60-75 minutes at room temperature as our standard.
7, Rinse again and apply a cover slip using an anti-fade coverslipping medium, we typically use Prolong Gold with DAPI in it to label the nuclei.
8. Cross your fingers and look in the microscope.
There are other things that can be done to reduce autofluorescence as well, such as doing sudan black counterstaining after immunolabeling, but the most important thing to do first is get the autofluorescence as low as possible to begin with.
Another consideration that could be very important for your staining since you are using murine tumor tissue is that your tissue will likely have alot of endogenous mouse antibodies in it that are present in the extracellular fluid bathing the tissue in vivo. If you need to use a primary antibody raised in mouse, your fluorescent secondary anti-mouse will bind not only to your specific primary but also will bind to the endogenous mouse antibodies in the tissue. There are mouse-on-mouse staining kits available to help block the endogenous mouse antibodies in the tissue before applying your primary antibody. (we don't have any real problems with this in our nervous system samples because thay are immune privileged and endogenous antibody levels are neglible.
Hope this helps. Let me know if you have questions (e-mail is the most reliable way to get hold of me: [email protected]). Go Gators! (I graduated from the Neuroscience department at UF a few eons ago. I hope your graduate experience in Gainesville is as good as mine was.)
Dave Sherry
Veronica,
Many paraffin embedded specimens have a very high level of autofluorescence. I have attached a protocol for immunolabeling paraffin sections that works pretty well for my lab. It has a lot of technical detail in it that I've left out of the discussion below. We work on brain and retina mostly, but we've used this procedure for other tissues as well. Our experience is that autofluorescence is also wavelength-dependent with shorter wavelength channels being worse than longer wavelength channels (Blue>> green> red> deep red). You might be able to help yourself by avoiding the high background channels when possible.
There are several things that you don't mention, so I assume there are important things that you are probably not doing in your labeling procedure.
1. A critical first step to immunofluorescent labeling of paraffin sections is to get the paraffin out of the tissue. This will expose the antigen and allow your antibodies to penetrate better. Triton alone probably won't do the job on paraffin sections. This is typically done using xylene, followed by rinses in ethanol and then exchanging to water or other aqueous buffer depending on what the next step in your procedure is. Even after removing the wax, we still see VERY high autofluorescence levels, so we do an autofluorescence reduction treatment.
2. Autofluorescence reduction. We routinely treat our sections with 1% NaBH4, which breaks double bonds and helps reduce autofluorescence. There are other procedures out there, this is just one option. The protocol has the details and the recipe. An important note here is that NaBH4 should be dissolved in WATER not PBS, which will cause it to bubble violently and destroy your tissue. NaBH4 will generate hydrogen gas, so some care in handlinh is a good idea. If NaBH4 doesn't work very well for you, you should try another one.
3. Fixation and paraffin embedding often denatures antigens, rendering them hard to detect with your antibodies. You are likely to need an antigen retrieval step. Again there are a variety of procedures out there, and you may need to try several to find the one that is optimal for you. Our standard approach to antigen retrieval is to treat the deparaffinized sections in hot citrate buffer (pH 6, 95 degrees). The protocol has the details and the recipe.
4. Following antigen retrieval most procedures have a blocking step that will use normal serum or bovine serum albumin to block sites that antibodies might bind to no-specifically. These solutions typically have some detergent in them to help open up the tissue and expose the antigen. The blocking solution also can be used as the buffer to dilute your antibodies.
5. The primary antibodies are applied. You will need to work out the proper incubation length and antibody concentrations for your specific situation. Our standard is to incubate the sections in primary antibody overnight at room temperature, but that can be adjusted according to need.
6. Rinse the sections to remove unbound primary antibody and apply fluorescent secondary antibody. We use 60-75 minutes at room temperature as our standard.
7, Rinse again and apply a cover slip using an anti-fade coverslipping medium, we typically use Prolong Gold with DAPI in it to label the nuclei.
8. Cross your fingers and look in the microscope.
There are other things that can be done to reduce autofluorescence as well, such as doing sudan black counterstaining after immunolabeling, but the most important thing to do first is get the autofluorescence as low as possible to begin with.
Another consideration that could be very important for your staining since you are using murine tumor tissue is that your tissue will likely have alot of endogenous mouse antibodies in it that are present in the extracellular fluid bathing the tissue in vivo. If you need to use a primary antibody raised in mouse, your fluorescent secondary anti-mouse will bind not only to your specific primary but also will bind to the endogenous mouse antibodies in the tissue. There are mouse-on-mouse staining kits available to help block the endogenous mouse antibodies in the tissue before applying your primary antibody. (we don't have any real problems with this in our nervous system samples because thay are immune privileged and endogenous antibody levels are neglible.
Hope this helps. Let me know if you have questions (e-mail is the most reliable way to get hold of me: [email protected]). Go Gators! (I graduated from the Neuroscience department at UF a few eons ago. I hope your graduate experience in Gainesville is as good as mine was.)
Dave Sherry
You could try a 5 min incubation with 1% evans blue. That should quench your autofluorescence in the green channel. Regarding your idea of using alexa 594 conjugated antibodies, I cannot tell. You would expect your tissue to have some fluorescence in the reds after evans blue. However, I have not tried the 594 and related spectra with evans.
If you decide to give it a try, please let us know your results!
Best,
Daniel
Fantastic answers, thank you so much! I will try these and get back to you.
I would agree with 1% NaBH4 quenching before secondary antibody labeling.
Try make serial dilution of your antibody then choice the best positivity / little background staining.
Regards
Ayman
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