Methods and Techniques Discussion => Image Analysis => Topic started by: RBallesteros on September 03, 2011, 06:52:42 AM

Title: dealing with autofluorescence
Post by: RBallesteros on September 03, 2011, 06:52:42 AM
Hi everyone!!

I'm new working with immunohistochemestry, particularly I'm working with ovarian tissue and I have realized that its autofluorescence is present across the whole wavelength since DAPI to Rhodamine. is there any way to cut out the whole background produced by the by the autofluorescence?

The software I'm using to analyze the images is Fiji

thank you very much to everyone how can put a bit of knowledge!!!
Title: Re: dealing with autofluorescence
Post by: gula on September 04, 2011, 07:41:44 AM
Please tell us, what kind of tissue preparation who have used.
unfixed - fixed - frozen - paraffin embedded?

what kind of controls have you performed to confirm, that it is really autofluorescence and not unspecific staining due to antibodies?

gula
Title: Re: dealing with autofluorescence
Post by: RBallesteros on September 05, 2011, 01:53:02 PM
Hi Gula

Thank you very much for your help!!

basically the tissue was fixed in 4% paraformaldehyde and embedded in paraffin. the sections are 8 micrometers thick.
the fluoroform of the secondary antibody that I used was alexafluor 488, the control was putting only the secondary antibody after the whole process of de-paraffin, rehydration, antigen retrieval and block, I saw fluorescence in the filter for GFP but also for Cy5, Rhodamine and DAPI.

In my sample with the primary antibody I saw higher fluorescence in GFP but the same fluorescence than my control in Cy5 Rhodamine and DAPI.

In addition I took another slide just after sectioning  (that wasn't dewaxed) and the tissue showed the same unspecific fluorescence across the all wavelength.

should I fix it with another thing? do I need to add another chemical in order to reduce the that fluorescence? which could be?

Thank you very much again!!
Title: Re: dealing with autofluorescence
Post by: Cardio on September 09, 2011, 12:10:56 PM
Hi Gula

Thank you very much for your help!!

basically the tissue was fixed in 4% paraformaldehyde and embedded in paraffin. the sections are 8 micrometers thick.
the fluoroform of the secondary antibody that I used was alexafluor 488, the control was putting only the secondary antibody after the whole process of de-paraffin, rehydration, antigen retrieval and block, I saw fluorescence in the filter for GFP but also for Cy5, Rhodamine and DAPI.

In my sample with the primary antibody I saw higher fluorescence in GFP but the same fluorescence than my control in Cy5 Rhodamine and DAPI.

In addition I took another slide just after sectioning  (that wasn't dewaxed) and the tissue showed the same unspecific fluorescence across the all wavelength.

should I fix it with another thing? do I need to add another chemical in order to reduce the that fluorescence? which could be?

Thank you very much again!!
Paraffin sections give higher autoflouresence and 4% pfa increases it as well. IF the cells are dieing then they will autoflouresce as well.

You can do IHC with DAB or your antibodies may be bad. Have you ever gotten to work before?
Title: Re: dealing with autofluorescence
Post by: RBallesteros on September 11, 2011, 07:10:39 AM
Hi Cardio Thank you very much for your answer.

I completely new working with immunohistochemestry and also in my lab, they are used to work only with single cells.

yesterday I have Inhibited endogenous peroxidase with 3% H2O2 in methanol for 15 minutes the autofluorescence was reduced a little bit specially in Cy5 filter at 200ms of exposure. should I increase the time in this step.

if the 4% PFA is increasing the autofluorescence, what can I use to fix the tissue?
now it is supposed I should not see anything in the different channels, if I do not stain with anything, am I right?

once again thank you very much for your help
Title: Re: dealing with autofluorescence
Post by: Cardio on September 12, 2011, 02:24:44 PM
Hi Cardio Thank you very much for your answer.

I completely new working with immunohistochemestry and also in my lab, they are used to work only with single cells.

yesterday I have Inhibited endogenous peroxidase with 3% H2O2 in methanol for 15 minutes the autofluorescence was reduced a little bit specially in Cy5 filter at 200ms of exposure. should I increase the time in this step.

if the 4% PFA is increasing the autofluorescence, what can I use to fix the tissue?
now it is supposed I should not see anything in the different channels, if I do not stain with anything, am I right?

once again thank you very much for your help

You are doing IF and my suggestion is to do IHC with DAB which is different. Look it up so you know the conditions and the differences. http://www.vectorlabs.com/catalog.aspx?prodID=33
Title: Re: dealing with autofluorescence
Post by: RBallesteros on September 14, 2011, 09:14:27 AM
Hi Cardio

Thank you very much for the lick I will try it on friday, and I will tell how is it working

ones again thank you very much