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Author Topic: IHC in long term formalin fixed paraffin-embedded Human Brain tissue  (Read 9540 times)

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Offline MiloDub11

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Hi everyone,

I've been trying to detect by IHC the pathogenic prion protein(using 3F4 monoclonal antibody) in brain tissue samples with more than 8 years of been submerged in formalin. I've been doing antigen retrieval by autoclaving the slides in citric acid buffer pH:6.0 at 121C during 30 minutes, then I treated with formic acid 10 minutes and 2hour in 4 M guanidine thiocyanate  and after all I am having no signal. I also tried to detect Glial Fibrilary Acidic Protein(GFAP) which is very common to be increased in Neurodegenerative diseases and because of this It would be expected to be found in my samples, but again no signal. That makes me wonder, If I am having no signal because of long term fixation instead because the sample is positive for pathogenic PrP. Have anyone hear about this problem before? or have anyone tried or achieved the detection of any protein in such samples? I hope you could give me some suggestions?

Thank you


Offline funk.106

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Re: IHC in long term formalin fixed paraffin-embedded Human Brain tissue
« Reply #1 on: March 19, 2009, 05:19:37 PM »
I was having issues trying to get tau5 signal, which stains for the protein in neurofibrillary tangles in Alzheimer's Disease, from my paraffin-embedded hippocampus slices from AD patients, and I finally got it to work using a ProK enzyme treatment. Im not sure if it will work for your staining or not, but you can try it:
•Heat citrate buffer (10 mM Citric Acid (anhydrous), 0.5% Tween-20, pH 6) to boiling in a glass beaker in the microwave. Add to coplin jar with slides to be immunostained. Place lid on top of coplin jar (do not screw on) and microwave on low power for 20 minutes (Can be adjusted) checking the liquid level in the jar every few minutes and add more if necessary to be sure that the tissue slices are thoroughly covered. (Be careful at this step bc a lot of pressure builds up under the lid and sometimes causes it to pop off and release a lot of liquid all at once, so the level drops immediately) While the tissue is microwaving, pre-warm humidified incubation chamber at 37°C.  Let liquid/tissue cool slowly at room temp for 10-15 minutes.
•Remove pre-warmed incubation chamber. Wipe off extra liquid using kimwipe and q-tip without touching tissue and set on incubation rack. Quickly and carefully outline the tissue with PAP pen. Apply enough TE-CaCl2-ProK solution (50 mM Tris, 1 mM EDTA, 5 mM CaCl2, 0.5% Triton-X 100, 20 ug/ml ProK---The Ca activates proK, so if the tissue is not well fixed, this should be eliminated) to tissue to completely cover it but without breaking the surface tension causing it to spill off. 250-300 µl per section should be enough. This must be done quickly so that the tissue does not become dry. Carefully put box in 37°C incubator for 10 minutes (Can be adjusted), then remove box and let cool at room temp for 10 minutes.
•Rinse sections in PBST for 2x5 minutes, then once in PB. Then continue on to peroxidase quenching or blocking.

Re: IHC in long term formalin fixed paraffin-embedded Human Brain tissue
« Reply #1 on: March 19, 2009, 05:19:37 PM »