Author Topic: Help with tunel assay  (Read 12178 times)

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Offline jgmatos

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Help with tunel assay
« on: November 17, 2010, 01:58:50 PM »
Hello there,

I'm starting to use the tunel assay in my lab, but on the first try we got a heavy background staining. We're using the roche kit, which has the converter reagent do work with DAB.
We tried pepsin, and citrate buffer pH 6, with microwave, and all slides got the same heavy background (although the citrate one was a little better).

Does anyone have an advice on what to tackle? pretreatment? Dilute the converter solution?

Notes: We did endogenous peroxidase blocking prior to adding the converter.
          We think our pepsin isn't nuclease free, which the kit says to use.

A very big thanks in advance

Help with tunel assay
« on: November 17, 2010, 01:58:50 PM »

Offline richard03

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Re: Help with tunel assay
« Reply #1 on: November 18, 2010, 10:54:46 AM »
Roche kit usually gives high background. It is most likely due to endogenous biotin (not peroxidase).

Millipore (Chemicon) kit works much better.
http://www.ihcworld.com/_protocols/apoptosis/apoptag-millipore-s7100.htm

Also proteinase k digestion method is better than citrate buffer method.

Offline jgmatos

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Re: Help with tunel assay
« Reply #2 on: November 18, 2010, 11:56:44 AM »
Thanks for your response.

I'm quite unexperienced with this kind of essay, but i already went twice through the datasheet of the roche kit and i found no mention of biotin. Has the kit changed? My cat. reference is: 11 684 817 910
Although they mention we can use bovine normal serum to block unspecific staining from the anti-fitc (DAB converter), so perhaps it has more to do with this antibody than with the biotin, no?

Also, i colleague of mine that used to use with essay told me i could dilute the converter antibody to reduce the background, does anyone have any experience with that?

Also, this essay is for research purpose, so our lab, at least in the near future, won't have acess to the milipore kit.

Again, thank your very much for your time. :)

Offline hardrob

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Re: Help with tunel assay
« Reply #3 on: November 22, 2010, 04:25:59 AM »
i dilute the tunel- probe 1/1 with buffer and the converter 3,5ml with 1,5ml buffer.if i don't dilute, i get enormous background.

after deparaffination preheat the slides 2 min in deionised water at 700 watt MW, and directly in protease( sigma/from strepto myceus) 0,15g/100ml pbs buffer for another 2 minutes.then rinse thoroughly with buffer.

works perfectly in my lab

all the best
« Last Edit: November 22, 2010, 04:28:23 AM by hardrob »

Offline jgmatos

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Re: Help with tunel assay
« Reply #4 on: November 24, 2010, 11:35:18 AM »
i dilute the tunel- probe 1/1 with buffer and the converter 3,5ml with 1,5ml buffer.if i don't dilute, i get enormous background.

after deparaffination preheat the slides 2 min in deionised water at 700 watt MW, and directly in protease( sigma/from strepto myceus) 0,15g/100ml pbs buffer for another 2 minutes.then rinse thoroughly with buffer.

works perfectly in my lab

all the best

Ok, i've been able to reduce the background by diluting the converter, and with Proteinase K (30'min) as my digestion.
Although i'm still far from the images of what it should look like.

Can you give me the full protocol? I'd appreciate it a lot.

Thanks in advance

Offline hardrob

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Re: Help with tunel assay
« Reply #5 on: November 29, 2010, 04:40:41 AM »
alright, here's my protocol
Deparaffination   :   Xylene:            2x5 min
         Ethanol:100      2x5 min
         Ethanol:96         5 min
         Ethanol:70         5 min
         Aqua dest         2 times



Antigen-Retrieval:   Aqua dest.:    Mikrowave    (600W)   2 min  (=ca. 60-75°C)
         
Enzyme:  Protease,,Sigma‘‘ (0,05 %) in PBS   2  min        37°C



Wash   :      Aqua dest         rinse
         Ethanol: 70 %         0,5min- to remove the enzyme completely
         Aqua dest         rinse

Block:         H2O2 (5%) diluted with  A.dest      10 min


Tunel Kit:      Boehringer 1:1 with  PBS      120 min       37C°
               


Wash   :      PBS Puffer          2 x


Label Enzyme-Complex:   anti Fluoreszin     (+ 1,5 ml Buffer)   30 min         37°C
      
         
Wash   :      PBS Puffer         2 x


Chromogenkomplex:   DAB    (Biogenex)      3 min

Rinse:         Aqua dest.


Counterstain:      Hämatoxilin Mayer      30 sec
         running Tapwater      5 min

Mounting

in my opinion ,,your'' proteinase k time is way to long and will cause massive background and also a loss in morphology. in ,,my'' protocol you i only need 2 minutes in protease, but don't forget to preheat in the mw.
by the way: when you read ,,Aqua dest'' in my protocol, it means ,,demineralized water''.you don't need de-ionized water.

godd luck and all the best.

Offline jgmatos

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Re: Help with tunel assay
« Reply #6 on: December 01, 2010, 02:16:08 PM »
Ok, thanks a lot. I've just finished my first sucessful, or at least readable slides, although i'm interested in using/adapting your protocol, since they aren't 100% perfect.

After that i'll post here my final protocol for others, that might need some help too.

Thanks to all.  :)

Re: Help with tunel assay
« Reply #6 on: December 01, 2010, 02:16:08 PM »