Dear researchgaters,
I have a question regarding staining protocols for oxidative tissue markers via IHC (4-HNE, MDA-2, 8-0HdG etc). I have found that these markers work in cryo stored material, but I have not had any success getting them to work on paraffin material. When staining with cryo, due to the poor freezing method of the brain tissue, I often have poor quality stains, high background and poor morphology on some many aspects of cells or tissue. As the tissue for paraffin is superb in terms of quality and structure, it would be ideal to get these markers to work on this tissue.
For cryo staining I:
Acetone fix 10 min, air dry 10 min, PBS soak 10 minutes, antibody block (10%fcs + TBS or Dako) for 1/2 h -> Primary antibody overnight at 4c.
Some protocols I have seen where people stain parrafin material they are not using a retrieve step but are using .1% triton in their blocks. I have tried this during EDTA retrieve but no luck. I have tried Citrate/EDTA retrieve, and also proteinase K. Using the EDTA/Citrate retrieve I get very beautiful staining of p22phox, iNOS, SOD2, SOD1 on the parrafin embedded tissue. Can someone please help me out here!
Unfortunately there is no ability to replicate these studies as they are primate brains and are from older projects. All of the brains at necropsy were placed in tins and put into liquid nitrogen so from that aspect are snap frozen. There seems to be a lot of variability though with how they are frozen and the quality of material. The paraffin brains are all quite nice though and have no issues.
Brain will be removed and dehydrated in graded serial of alcohol and embedded in paraffin wax .Five micrometer thick sections were cut and stained by hematoxylin and eosin (H&E).
Hi Jordan, put a piece of brain (1 x 1 x1 cm in size) in ice cold 4%PFA or 10 % Formalin, what you'llget in your lab. Lieve it in the fixativ a couple of days in the fridge. While the tissue is thawing the formalin fixation starts. After the fixation process the brain tissue as Jalpa recommaded it. The H&E stain will help you to get an impression of the quality of the tissue. Positiv controls for immunohistichemistry will help to find out weather the reason for the negativ result is the tissue processing, the IHC protocol or the antibody by itself. To establish new antibodies or new protocols under new circumstances it is also helpfull to run a delution raw to optimize the antybody concentration. Good luck!
Thank you Jalpa and Ute!
Do you have any recommendations on why an antibody would work mainly on cryo and not as well on paraffin?
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