[Histonet] TUNEL on FIXED rat PUPS

Liz Chlipala liz <@t> premierlab.com
Tue Dec 16 17:15:03 CST 2008


We have not used that kit specifically we use the one from Roche but we get fine results with the following fixatives, 10% NBF, 4% Paraformaldehyde and Davidisons, its also works fixed and EDTA decalcified samples.  You will need to add a proteinase K step to your protocol.  You need to order a specific proteinase K the one you may use for IHC may not work.  We get ours from Roche here is the catalog number and how we prepare the stock and working solutions, you may need to titer the concentration of proteinase K for your particular project we use it at a concentration of 20ug/ml.   I have also added our basic protocol so you can see where the protienase K step figures in.

Proteinase K, recombinant, PCR Grade Roche Applied Science      03 115 879 001

Proteinase K Stock Solution - 20mg/ml

Proteinase K                    1 vial - 100mg
Distilled water                 5 mls  

Once the proteinase K lyophilized enzyme has been reconstituted with distilled water then aliquot into
RNase free 0.6 ml microtubes a volume of 20 to 40µls and store at -15 to -25°C. 

Shelf Life:     12 months
Storage:         -15 to -25°C

Proteinase K Working Solution - 20ug/ml

For each slide that needs to be stained you will need to prepare a minimum of 125µls of proteinase K working solution.  100µls of working solution will be placed on each slide.  The working solution is prepared as follows:

Proteinase K Stock Solution     10µls
10Mm TRIS/HCL pH8.0             990µls

Make fresh prior to use.  Remove one aliquot vial of the stock 20mg/ml proteinase K from the freezer
and thaw.  Unused stock proteinase K may be stored in the refrigerator for 7 days, then discard.

Shelf Life:     make fresh prior to use, discard unused portion
Storage:        NA


When performing the TUNEL stain the maximum number of slides that can be run at a time is 12.  Within
the 12 slides, 10 will be test samples, 1 is a positive control (DNase treated) and 1 is a negative control
(Label Solution without Enzyme Solution).  All but the rinse steps are performed either in a humidity chamber or in the Dako Hybridizer.

1.      Xylene                                                      5 minutes
2.      Xylene                                                      5 minutes
3.      100% alcohol                                            2 minutes
4.      100% alcohol                                            2 minutes
5.      95% alcohol                                                 1 minute
6.      95% alcohol                                             1 minute
7.      Tap water rinse                                         1 minute
8.      Distilled water rinse                                           1 minute
9.      PBS pH7.4       0.1% tween rinse                                1 minute or more
10.     Prepare working proteinase K - see solutions
11.     Working proteinase K (75 -200µls/slide)                 30 minutes
12.     PBS pH7.4 0.1% tween rinse                              3 minutes

13.     PBS pH7.4 0.1% tween rinse                              3 minutes
14.     Prepare DNase1 working solution - see solutions
15.     DNase1 (75 -150µls/slide)                                       10 minutes

Note:  apply DNase1 to only the positive control sample, the rest
of the samples will remain in PBS pH7.4 0.1% tween buffer

16.     PBS pH7.4 0.1% tween rinse                              3 minutes
17.     PBS pH7.4 0.1% tween rinse                              3 minutes
18.     Prepare TUNEL reaction mixture

Note:  The TUNEL reaction mixture should be prepared immediately
before use.  Keep TUNEL reaction mixture on ice until use.  Unused
reagent may be stored at -70°C for future use.

Prepare the TUNEL reaction mixture and negative control solution as follows:

a.      The TUNEL kit reagents are stored in the -70°C freezer
b.      Remove one vial 1: enzyme solution and one vial 2: label solution
from the freezer
c.      Remove 100µl of the label solution and place it into a microtube this
solution will serve as the negative control solution
d.      Add the total volume of vial 1: enzyme solution (50µl) to the remaining
450µl in vial 2: label solution and mix well

19.     Remove slides from PBS and dry area around sample
20.     Add 50 to 100µl of TUNEL reaction mixture
21.     Incubate at 37°C in the Dako Hybridizer                         60 minutes
NOTE:  for the negative control add 50 to 75µl of the label solution to
each sample.  If necessary to ensure a homogenous spread of TUNEL
reaction mixture across the sample and to avoid evaporative loss, the
samples can be covered with a coverslip during incubation.
22.     PBS pH7.4 0.1% tween rinse                              3 minutes
23.     PBS pH7.4 0.1% tween rinse                              3 minutes
24.     PBS pH7.4 0.1% tween rinse                              1 minute
25.     Add 50 to 100µl of Converter-AP (vial 3) to each sample
26.     Incubate at 37°C in the Dako Hybridizer           30 minutes

NOTE:  If necessary to ensure a homogenous spread of TUNEL
reaction mixture across the sample and to avoid evaporative loss, the
samples can be covered with a coverslip during incubation.

27.     PBS pH7.4 0.1% tween rinse                              3 minutes or more
28.     PBS pH7.4 0.1% tween rinse                              3 minutes or more
29.     Wash Buffer                                             1 minute
30.     Prepare Vulcan Fast Red Substrate Solution

To 2.5 mls of Vulcan Fast Red Buffer add one drop of vial A and one
drop of vial b.  Use immediately, discard any leftover reagent.

31.     Vulcan Fast Red Substrate Solution                              10 - 30 minutes

32.     Distilled water                                         2 minutes
33.     Distilled water                                         2 minutes
34.     Hematoxylin                                             3 minutes
35.     Distilled water                                         1 minute
36.     Wash Buffer                                             1 minute
37.     Distilled water                                         1 minute
38.     Let slides air dry
39.     Xylene                                                  1 minute
40.     Mount and coverslip
41.     Review positive and negative control slides for appropriate staining

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
P.O. Box 18592
Boulder, CO 80308
phone (303) 682-3949
fax (303) 682-9060
liz <@t> premierlab.com

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Premier Laboratory, LLC
1567 Skyway Drive
Unit E
Longmont, CO 80504

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Guillermo Palchik
Sent: Tuesday, December 16, 2008 3:37 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] TUNEL on FIXED rat PUPS

Dear Histologists...

I am currently doing an experiment in which I perform TUNEL on brain slices that were treated with certain drugs, to examine the levels of apoptotic cell death. The rat's brain is flash frozen (scooped from the skull, directly into cold isopentane) air dried, and then stored into a -80 C freezer.  We then cut the brain into 20 um slices using a cryostat and mount them on slides, and store them in a -20 C freezer, where they remain until the TUNEL step. The TUNEL is performed using the Apoptag Plus kit from Chemicon using Peroxidase/DAB (Cat. # S7101) and counterstaining with 0.5% methyl green. This has been done in the lab for quite some time now and we are able to get results... We want to switch over to perfused brains (instead of flash frozen). 
However, we have had ZERO positive staining once the brains are fixed (in 4% PF). This has been corroborated by other lab members that have tried it for a couple of years already... The protocol that came with the kit has a section for flash frozen and a section for fixed tissue, and since I have done TUNEL using fixed tissue before, I know that it is possible to do TUNEL in fixed tissue, however we cannot get any positive staining whatsoever... Along these lines, since the first step of the Apoptag TUNEL protocol is to fix the tissue with 4% PF (for 10 min), this has led me to believe that the problem is indeed the initial fixation with PF (at the time of perfusion).
I should say that we work with rat PUPS for this and that the immature brain is not the same as the mature brain (the immature brain has more fat, for example) and that this might be the cause of the problem...In any case, I was doing some research and I wanted to try using Zinc Formalin as a perfusate and see if this would allow us to do the TUNEL.

I would appreciate any comments and suggestions regarding this. I am sorry for the lengthy email, but I wanted to show a more or less complete picture, in case I am overlooking other factors...

Guillermo Palchik
gp62 <@t> georgetown.edu

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