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Antigen retrieval


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#1 Rajamani

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Posted 20 January 2011 - 09:04 PM

I am doing antigen retrieval on brain sections. Antigen retrieval buffer is tris-Hcl buffer pH 9.5. Heating the buffer at 80 C for 30mins and cool it for 1hr and then process it for regular immunocytochemistry. I use polyclonal primary antibody, biotinylated secondary antibody and incubate with ABC complex and later on stain with glucose oxidase Ni DAB solution. I use free floating sections for the assay but I have trouble when I mount these sections, there are lots of wrinkles and sometimes some parts are missing. As far as staining goes, I get a clear staining. Do you have any suggestions for this? Have any of you tried antigen retrieval on slide mounted sections?? IF so what would be your coating of the slides. I use gelatin coated slides and didnt work well. All sections got detached from it.

#2 Rsm

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Posted 21 January 2011 - 12:21 AM

I made AR on frozen sections on poly-Lysine coated slides. Some fall off, that happens. You need to dry them extensively (1h using a fan or O/N), that helps. Gelatin doesn't work as well, that's true. So cut sections, put on slide, dry, make AR.

rsm
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#3 Rajamani

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Posted 21 January 2011 - 11:14 AM

Thanks a lot.

can you gimee the protocol for preparation of lysine coated slides. What do u mean by drying them extensively, the step to be done after AR or before AR.

Thanks

I made AR on frozen sections on poly-Lysine coated slides. Some fall off, that happens. You need to dry them extensively (1h using a fan or O/N), that helps. Gelatin doesn't work as well, that's true. So cut sections, put on slide, dry, make AR.

rsm



#4 Rsm

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Posted 24 January 2011 - 12:25 AM

Sorry, we buy the coated slides :rolleyes:. But there are lots of protocols online, just google for it.
The drying is after cutting and before AR.
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#5 beth

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Posted 26 January 2011 - 07:46 AM

You can buy a bottle of poly-L-lysine, which will coat a TON of slides. Using a pastuer pipette, put one drop on a slide. Get another slide and put on top, squishing the liquid to cover both slides. Leave the slides wet side up on some tissue until dry. You can do a whole load at once and store. We put a little pencil mark on the top left corner of the frosted part of the slide, so we know which side of the slide is coated (also stops you accidentially wiping off your sections later!).

When you cut the sections, are you floating them on warm water before mounting on the slides? This will really help get rid of any wrinkles.

We always leave the slides to dry AT LEAST overnight in a drying oven before doing anything else to them. I've never had any sections fall off when doing antigen retrieval after this.

Good luck




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