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Author Topic: cytospin and forzen sections  (Read 7801 times)

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Offline braz

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cytospin and forzen sections
« on: November 17, 2005, 08:57:37 PM »
Hi!
I've been having bad results with immunostaining for activin receptors. I'm working with some frozen tissue sections (in OCT) and some cytospin slides; both fixed in cold acetone. Using the standard avidin-biotin (DAB)technique, I've had prominent edge effect in both, with no staining in the center of the sample.
I'm using the primary Ab in the concentrations suggested by the manufacturer, which are 7 times greater than the ones used by other research groups for paraffin sections. Can that have any implication in the results I'm having?

cytospin and forzen sections
« on: November 17, 2005, 08:57:37 PM »

Offline ImmunoNYC

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Re: cytospin and forzen sections
« Reply #1 on: November 18, 2005, 12:13:31 AM »
I would definitely titrate the primary to find the best concentration for the tissue prep and secondary antibodies. Every lab is different and often times different concentrations are needed. Try say 2 or 3 dilutions above and below the suggested concentration. So if they recommend you use 1 ug/ml try 0.1, 0.3, 1, 3 and 10 ug/ml or something like that.

Also are you sure all your reagents are "good". That your primary is good, secondaries are specific etc. Do you have a control tissue which you KNOW should be positive.
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Offline ImmunoNYC

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Re: cytospin and forzen sections
« Reply #2 on: November 18, 2005, 12:14:36 AM »
Also since you are having edge effect are you taking steps to make sure your samples do not dry out?[/color]

Quote from: "braz"
I've had prominent edge effect in both, with no staining in the center of the sample.

Offline braz

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Re: cytospin and forzen sections
« Reply #3 on: November 18, 2005, 12:38:23 AM »
First of all, thanks for the reply. I appreciate your help.
Well, regarding dilutions, I tried one below the recommended (half of it) and had no result. I'll try different concentrations next.
The reagents are good and pretty new. The secondary AB match the primary and everything works, because I ran IHC for another antigen at the same time, using the same staining protocol and the same reagents and it came out perfect. The differences were the primary AB and the tissue (in this case, I had paraffin sections).
The samples I used as a positive control are extensively described in literature as positive, but in paraffin. I didn't find any report of staining for these receptors in frozen tissue. But I thought that shouldn't be a big problem... I guess I was wrong...  :wink:

Offline braz

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Re: cytospin and forzen sections
« Reply #4 on: November 18, 2005, 12:45:55 AM »
Oh, and I'm quite sure they didn't dry.
Anyway, I'll keep trying and looking for help. Comments and suggestions are welcome!! :wink:

Re: cytospin and forzen sections
« Reply #4 on: November 18, 2005, 12:45:55 AM »