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Author Topic: Autoradiography on tissue sections  (Read 18818 times)

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Offline funk.106

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Autoradiography on tissue sections
« on: November 17, 2010, 09:32:40 AM »
Hi!
My boss is wanting me to attempt some autoradiography on tissue sections, which I have never done. It will be a tritiated small molecule that will be custom made (ie super expensive) and precious human tissue samples, so I'd rather not waste more than necessary by trying to learn what I'm doing. I have experience with IHC and fluorohistology, and I am wondering if it is generally the same procedure, except for the radioactivity, of course, and the method of development. I have been reading Rogers' Techniques of autoradiography to try to learn more about it, but the book is quite old, and I was wondering if anyone had any advice either by first-hand experience about the key differences/similarities between protocols of general histology/IHC and autoradiography, or had suggestions for a more recent manual of autoradiographic techniques.
I appreciate any suggestions!!
« Last Edit: November 17, 2010, 09:34:19 AM by funk.106 »

Autoradiography on tissue sections
« on: November 17, 2010, 09:32:40 AM »

Offline rmcgandara

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Re: Autoradiography on tissue sections
« Reply #1 on: November 17, 2010, 10:16:18 AM »
The technique itself is old and not much has change.

This is the protocol I have used for intestinal samples:


1) Dewax slide overnight in Xylene
2) Turn on the Churchill cooling plates one hour before dipping commences
3) Rehydrating down a graded series of alcohols to distilled water 3x 5min.
4) Place into distilled water containing Kodak Photo-Flo (200 ml dH2O + 6 drops Photo-Flo)
The actual dipping process takes place in the darkroom under safelight conditions.
5) The emulsion used is, which comes as gelatinous shreds in light-tight boxes. 10 ml of distilled water should be pre-heated to 42C in the Dri-Block. 10 ml of Ilford K5 emulsion are then added to this (under safelight conditions). Gently even the vial to mix the emulsion and water, being careful not to get any bubbles into the solution. Keep the vial in the Dri-Block to enable the emulsion to melt, which will take approximately 15 minutes. When the emulsion has melted it should be poured into the dipping jar in the Dri-Block.
6) Coat the slides with emulsion and laid them out on the cooling plates and allow them to dry at 20C for 3 hours.
7) When fully dry, the slides should be placed in double light-tight boxes containing a small amount of Silica Gel. The slides are now left to expose at 4C in the fridge.

8) After use, the dipping jar should be immediately washed out with hot running water, and then placed in the Hydrochloric Add bath in the fume cupboard overnight It can then be rinsed under running tap water and placed in the Decon bath (also in the fume cupboard) for a few hours. After this time it should then be rinsed again under running tap water, allowed to dry, and then returned back to the drawer.






The developing process takes place in the darkroom under safelight conditions.

1) Place the slides in Kodak D19 Developer for 8 minutes at 20C 0.5C (100ml D19 + 100ml dH2O).
2)Transfer them to a 1-2% Acetic Add Solution for 2 minutes at 18-22C.
3) After the slides have been in the acid, they are transferred to a dish of Ilford Hypam Fixer. The Fixer is diluted one part to five parts of distilled water, and the slides stay in the fixer (at 18-22C) for 6 minutes.
4) The slides should then be rinsed in running filtered and deionised water for at least 30 minutes.
6) Slides can be stained with Haematoxylin (1 min) and Eosin (2min)as normal.

Offline funk.106

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Re: Autoradiography on tissue sections
« Reply #2 on: November 17, 2010, 10:32:30 AM »
Thank you for your reply! Do you think that at the end of the autoradiography development, instead of doing the H&E staining as you have listed, it would be possible to do IHC staining? or have you ever tried anything like that?

Offline Cardio

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Re: Autoradiography on tissue sections
« Reply #3 on: November 17, 2010, 11:27:23 AM »
Thank you for your reply! Do you think that at the end of the autoradiography development, instead of doing the H&E staining as you have listed, it would be possible to do IHC staining? or have you ever tried anything like that?
So a couple of things here.
I do Autorads a lot and we dilute our emulsion one to one in DI water to save on money which could be an option for you.

To answer your question about IHC you can do it before the emulsion dip and the DAB stain will be retained.

Offline funk.106

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Re: Autoradiography on tissue sections
« Reply #4 on: November 17, 2010, 02:16:57 PM »
I do Autorads a lot and we dilute our emulsion one to one in DI water to save on money which could be an option for you.

Thanks for the suggestion! One concern that we have for the set-up is that there may be low binding stoichiometry, thus giving us a low signal. We want to use a tritiated compound so that we have better resolution and are able to see pathology morphology, though. For this type of situation, would it be better to use undiluted emulsion, or does it not work like that?

Offline Cardio

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Re: Autoradiography on tissue sections
« Reply #5 on: November 18, 2010, 03:59:55 PM »
I do Autorads a lot and we dilute our emulsion one to one in DI water to save on money which could be an option for you.

Thanks for the suggestion! One concern that we have for the set-up is that there may be low binding stoichiometry, thus giving us a low signal. We want to use a tritiated compound so that we have better resolution and are able to see pathology morphology, though. For this type of situation, would it be better to use undiluted emulsion, or does it not work like that?

So it sounds like you are using the small molecule as a radioactive probe for something specific in human tissue. Is this correct? The common use for emulsion in my lab is the simple DNA synthesis with tritated thymidine. You can imagine that you have a lot of incorporation into the DNA with a synthetic cell. The dilution is something that the company I believe Ilford recommended to us.

In your case I would have a positive control to test the probe on before I use the samples. YOu could also figure out the incubation time off of the controls. I will let you know that you will have to find out the incubation time experimentally. ITs not as simple as 1 day equals x amount of silver grains. I know it should be but when I have run the test and it doesn't play out that way for a number of reasons (thickness of the section, binding of the probe, thickness of the emulsion.

I would highly recommend also testing your emulsion before the dip. I have noticed certain background artifacts with certain lots. Meaning you can get a nonspecific clumps of silver grains do to the emulsion.

Also, if you do a general stain make sure it doesn't react with the emulsion. Some stains actually expose the emulsion. Example Toluidine Blue.
« Last Edit: November 18, 2010, 04:16:16 PM by Cardio »

Offline Cardio

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Re: Autoradiography on tissue sections
« Reply #6 on: November 18, 2010, 05:10:47 PM »
So to answer your question, thicker emulsion may lead a better sensitivity but you will also have a certain background level that will increase with the thickness. Diluting the emulsion may not be way to go for you but you would have to try it to know. You should aliquot it though in 50 ml falcon tubes and triple wrap it in tin foil. Also I don't use a safe light at all. Everything is done in the dark to keep the emulsion background low. By the way most university dark rooms are not really dark. People use those stupid glow in the dark stickers for their westerns and the revolving doors felt is often old and worn. All this affects the emuslions background.
« Last Edit: November 19, 2010, 11:07:11 AM by Cardio »

Offline funk.106

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Re: Autoradiography on tissue sections
« Reply #7 on: November 19, 2010, 11:38:23 AM »
Thanks! Yes, we're basically interested in seeing if a small molecule that we design will specifically bind in situ, so we're wanting to make it radioactive in order to detect it. I'm going to start by replicating what other people have done in similar situations, but since I'm new at this and it's a new probe/new set up, I'm sure there will be a lot of trial and error...and hopefully I can get a reasonable result sooner rather than later. I'll keep your tips in mind and hopefully get it figured out!

Offline Cardio

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Re: Autoradiography on tissue sections
« Reply #8 on: November 19, 2010, 03:05:51 PM »
I'll give you a few more tips.

1. A brown/orange safety light is better than a red safety light. red will work though.

2. For dipping you want to aliquot the emulsion into 50 ml falcon tubes and use the tubes for dipping the slides. No need to having a dipping jar because it waste the emulsion. Aliquot levels are based on what is covers your sections on the slides,,, see point 4.

3. Place the slides back to back to dip them (doesn't waste emulsion on the back side of the slides). Use cloths pins to clasp the slides to dip into the emulsion.

4. Sections can be placed on the bottom part  of the slides so you can use less emulsion in the tube to cover the sections.

5. IF you reheat emulsion for more dips the background level goes up with each reheating. In my hands it takes about 10 to 15 before the background level gets to high.
 
6. Store the slides in a slide rack in a ammo box. The boxes are cheap and easy to use.

7. Just a FYI, Dapi or Hoescht survives the dipping/fixation process and I know egfp survives too. In general the emulsion slightly enhances the autoflouresence.

Good luck

Offline rmcgandara

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Re: Autoradiography on tissue sections
« Reply #9 on: November 23, 2010, 08:47:03 AM »
Cardio

Really nice tips I wish I have had them 5 years ago :)

Do you reuse the emulsion? I have always used fresh emulsion because I thought reusing would increase the background greatly (and that would impact me because I have to count grains).

Also do you have an idea how long does the emulsion last? I know that they reckon a shelf life of a couple of months, but have you used it past that time?

We have a specially made glass dipping jar that fits the slide perfectly and allow us not to waste too much emulsion.

Cheers

R

Offline funk.106

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Re: Autoradiography on tissue sections
« Reply #10 on: November 23, 2010, 09:02:07 AM »
These are very good tips. Thank you!

Offline Cardio

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Re: Autoradiography on tissue sections
« Reply #11 on: November 23, 2010, 04:12:30 PM »
Cardio

Really nice tips I wish I have had them 5 years ago :)

Do you reuse the emulsion? I have always used fresh emulsion because I thought reusing would increase the background greatly (and that would impact me because I have to count grains).

Also do you have an idea how long does the emulsion last? I know that they reckon a shelf life of a couple of months, but have you used it past that time?

We have a specially made glass dipping jar that fits the slide perfectly and allow us not to waste too much emulsion.

Cheers

R


I resuse the emulsion about 10-15 times or when the background level becomes too high for my liking. The background is still really low at 5 times though.

As for the half life of emulsion it may very well depend on where you get it from. I believe there is only one supplier for fine grade emulsion in the U.S. and I know that emulsion will last at least 6 months to a year.

IF you ever break those jars just use a plastic 50 ml falcon tube and store your emulsion in that by wrapping tin foil around it.

Offline funk.106

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Re: Autoradiography on tissue sections
« Reply #12 on: February 17, 2011, 05:08:19 PM »
Hi again! The compound that we're having sythesized is close to being completed, so I'm trying to make sure that I have a good grip on what I'm going to try to do and I'm realizing that I'm confused about something--nuclear emulsions and films. When I was first thinking about this I thought that they were used together, but I think maybe I'm wrong about that? Specifically, I'm looking at the amersham hypercoat emulsion and amersham hyperfilm 3h, which from what I can tell are used mutually exclusively--the film is apposed with the uncoated slide and then developed and you have the film as your data, alternatively, the emulsion is coated onto the slide and then developed and then you can look at the developed silver along with the tissue directly. To me it seems like the emulsion is easier and makes more sense if I am interested in the spatial distribution, but it seems like more people use the film. Can you help me understand why this would be?
Also, do you have any idea what the sensitivity of detection these have (ie, if western blots can detect ~10 nmol of protein, how many uCi can autoradiographic emulsions/films detect)?
« Last Edit: February 18, 2011, 02:39:19 PM by funk.106 »

Offline funk.106

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Re: Autoradiography on tissue sections
« Reply #13 on: February 22, 2011, 05:28:58 PM »
As for the half life of emulsion it may very well depend on where you get it from. I believe there is only one supplier for fine grade emulsion in the U.S. and I know that emulsion will last at least 6 months to a year.

Another question...what is your preferred emulsion? I have seen Amershams (which has been discontinued) and then Ilford and Kodak. Do you think one is better than the other? or is there another one available?

Offline Cardio

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Re: Autoradiography on tissue sections
« Reply #14 on: March 03, 2011, 11:28:03 AM »
Also, do you have any idea what the sensitivity of detection these have (ie, if western blots can detect ~10 nmol of protein, how many uCi can autoradiographic emulsions/films detect)?

So I have 0 experience with the films but I would look up the price of the films and compare that with the emulsion. I have a hunch that is the reason why more people are using the films. As far as the senstivity this might help. http://www.jstor.org/stable/pdfplus/3570804.pdf?acceptTC=true I would also contact Ilford who may point you in the right direction.
We use ilford emulsion (over a 1000 USD). One thing that I am confused about is why you used TH3 labeled compound. Why not label it with a Flag or HA unless of course those are too big. ALso one advantage you can have with using emulsion is that you will be able to use Hoescht or Dapi plus antibodies with the emulsion. The background flouresence increases but its not too bad with hoescht.
« Last Edit: March 03, 2011, 11:38:02 AM by Cardio »

Re: Autoradiography on tissue sections
« Reply #14 on: March 03, 2011, 11:28:03 AM »