IHC - No staining whatsoever - (Feb/14/2008 )
Hey All,
I'm having a real technical nightmare here and hoping someone can help. The problem is that I have tried several antibodies to do HRP staining (which I know are reliable), and have been unable to get any staining (background or otherwise) with any of them. Samples are as follows: Mouse tissues are collected and fixed immediately in 4%PFA for a couple of days. These are then place in 30% sucrose for 24 hours, frozen in OCT and cut at 80um on a cryostat. Prior to staining, they are acetone fixed for 5m at -20C and rehydrated. I've tried everything: 1% SDS, .05% trypsin, 0.1% triton, etc. I really can't heat them past ~50C as they begin to lose morphology and I actually am not even sure it's an antigen retrieval problem. Any suggestions would be immensely appreciated. Thanks!
Mike
I'm having a real technical nightmare here and hoping someone can help. The problem is that I have tried several antibodies to do HRP staining (which I know are reliable), and have been unable to get any staining (background or otherwise) with any of them. Samples are as follows: Mouse tissues are collected and fixed immediately in 4%PFA for a couple of days. These are then place in 30% sucrose for 24 hours, frozen in OCT and cut at 80um on a cryostat. Prior to staining, they are acetone fixed for 5m at -20C and rehydrated. I've tried everything: 1% SDS, .05% trypsin, 0.1% triton, etc. I really can't heat them past ~50C as they begin to lose morphology and I actually am not even sure it's an antigen retrieval problem. Any suggestions would be immensely appreciated. Thanks!
Mike
Is it a nuclear protein you are trying to stain for? Does your antibody have azide in it? (Azide inhibits HRP). Have any stains worked in your hands before?
I'm having a real technical nightmare here and hoping someone can help. The problem is that I have tried several antibodies to do HRP staining (which I know are reliable), and have been unable to get any staining (background or otherwise) with any of them. Samples are as follows: Mouse tissues are collected and fixed immediately in 4%PFA for a couple of days. These are then place in 30% sucrose for 24 hours, frozen in OCT and cut at 80um on a cryostat. Prior to staining, they are acetone fixed for 5m at -20C and rehydrated. I've tried everything: 1% SDS, .05% trypsin, 0.1% triton, etc. I really can't heat them past ~50C as they begin to lose morphology and I actually am not even sure it's an antigen retrieval problem. Any suggestions would be immensely appreciated. Thanks!
Mike
Is it a nuclear protein you are trying to stain for? Does your antibody have azide in it? (Azide inhibits HRP). Have any stains worked in your hands before?
Thanks for the reply. I have tried both membrane (PDGFR) and nuclear (Ki67) staining, neither of which have azide in them. We have had success with both these antibodies in parrifin embedded tissue microarrays. Could it be due to the thickness of the tissue slice?
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Thanks for the reply. I have tried both membrane (PDGFR) and nuclear (Ki67) staining, neither of which have azide in them. We have had success with both these antibodies in parrifin embedded tissue microarrays. Could it be due to the thickness of the tissue slice?
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No worries 
In my experiences I couldn't get nuclear stains to work in frozen tissues. I tried gentle antigen reteival and it never worked 
My paraffin embedded slides always worked though. Is anyone else in your lab doing IHC? Is it working for them? Could it be a prob with reagents? How thick are your tissue slices?
I'm just curious.... why do you fix in para and then freeze. Why not snap freeze straight away, which would work much better for cryo sections. Alternatively why not fix in para and then go through conventional wax processing especially when you know that this works. Cryo's tend to lose a bit of morphology as well.
You are so right. I missed that bit. I have never heard of fixing before frozen sections.
Thanks for the replies. The tissues are PFA fixed because they are bone and need to be decalcified (0.5M EDTA) for 6 days prior to sectioning. Our lab doesn't have access to a tape cryostat, although I have heard that that is the best way to preserve morphology. Sections are 80 um thick for experimental reasons (needle in a haystack kind of thing), so could it be that the antibody is not penetrating enough? I would assume that I would at least get superficial staining though...
aaahhh bones! When I have stained bones I have had to decalcify for ~ 2 weeks! And the method is different depending if you want frozen or paraffin. Also, why don't you just cut serial sections of your bones and therefore have thinner slices? That's what I did when "looking for the needle in the haystack".
Here's the protocol:
1. Fix in 4% PFA 4 hours, 4 deg.
2. Wash in PBS/2% sucrose 4deg for 2 days (whilst rocking). Change solution twice a day.
3. Wash in Tris.HCl (0.1M pH 6.5) for 24 h.
4. Decalcify with 0.1M Tris.HCl pH6.95 containing 7.5% polyvinylpyrolidone(PVP)-40T and 10% EDTA for 2 weeks at 4deg. Change the solution twice a week.
5. at this point bones can be placed in 70%EtOH and embedded in paraffin. If doing frozen sections, go to step 6.
6. When fully decalcified wash in Tris, 4deg 30 mins
7. Place in 20% sucrose 24 hours 4deg
8. Infiltrate in 20%sucrose and OCT (1:1), 24 hours, 4deg.
9. Infiltrate with 2.5% DMSO/20% sucrose and OCT (ie: 0.5ml DMSO, 25ml 20% sucrose, 25 ml OCT). ON 4deg.
10. Place bones in OCT and freeze!
This protocol came from a lab which did IHC on bone sections *every day*. Hope it helps!
I'm having a real technical nightmare here and hoping someone can help. The problem is that I have tried several antibodies to do HRP staining (which I know are reliable), and have been unable to get any staining (background or otherwise) with any of them. Samples are as follows: Mouse tissues are collected and fixed immediately in 4%PFA for a couple of days. These are then place in 30% sucrose for 24 hours, frozen in OCT and cut at 80um on a cryostat. Prior to staining, they are acetone fixed for 5m at -20C and rehydrated. I've tried everything: 1% SDS, .05% trypsin, 0.1% triton, etc. I really can't heat them past ~50C as they begin to lose morphology and I actually am not even sure it's an antigen retrieval problem. Any suggestions would be immensely appreciated. Thanks!
Mike
hallo,
i also had problems with my IHC staining and it takes me a year to establish the protocol. 1. the fixation (special fixation solution containing 2% formaldehyde, 5% acetic acid and 75% ethanol). also very critical is the fixation time, in my case it was 24h per mm3 tissue. 2. antigen retrieval: microwave heating 5min 600W, 15min 200W in citrate buffer. 3. incubation time and temperature of 1st AB could also be very critical. i let 1. AB over night at RT. 4. secondary antibody is not directly labelled with HRP it is biotinylated (Vecor) in combination with Vectastain ABC-Standard Elite Kit.(Vector).
hope that helped a bit,
i´m with you