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Author Topic: IF on frozen mouse tissue  (Read 10420 times)

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Offline anwaar

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IF on frozen mouse tissue
« on: May 27, 2005, 09:15:08 AM »
Hi everyone,
I am using frozen mouse tissue (Liver and Kidney) for IF. I take a piece of the tissue and freeze it in OCT on dry ice. Then I freeze in -80 till sectioning. After sectioning I do the following
1. Dry the slides for 30 min at room temp.
2. Fix in cold acetone for 30 min
3. Air dry for 30 min
4. stain with primary and secondary (Alexa 488) antobody using standard procedure (Blocking, washes etc).

The problem is that I dont see nice nuclei after staining with DAPI. Nuclei are rough and disformed. Also there is very strong back ground in FITC channel  (even with DAPI only).

Can somebody give me any suggestions how to deal with these problems. Thanks.
Anwaar

IF on frozen mouse tissue
« on: May 27, 2005, 09:15:08 AM »

Offline ImmunoNYC

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IF on frozen mouse tissue
« Reply #1 on: May 28, 2005, 06:06:19 PM »
I suggest you alter your freezing protocol to a more optimized protocol such as using isopentane/LN2 or isopentane/dry ice. You might need to speed up your freezing to preserve the morphology better.

In addition why not try what I do for my frozens which seems to work well ...  try to take your slides directly from the freezer into acetone for a quick fix of 3-5 minutes. Briefly dry and  do a post-fix in 10% NBF for 10 sec and then wash in PBS to remove OCT.

Other suggestions I have heard of but don't have personal experience with to improve mouse frozen morphology:

- Two acetone fixations separated by a brief drying.
- 75% acetone, 25% pure EtOH for 10 min
- 10 min submersion in 1% PFA and NO ACETONE (might affect your antigens)

Offline madeleine huey

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IF on frozen mouse tissue
« Reply #2 on: May 29, 2005, 09:46:49 PM »
I have done hundreds and thousands of frozen mouse tissue, and work for me very well.   Here's my suggestion;
1) Put tissue in OCT & freeze immediatly in Ethanol/dry ice (put dry ice in 100% ETOH), until OCT turn solid white color (store OCT in -70C until ready to cut)
2)If tissue are store in freezer, then thaw & dry tissue section from freezer to room temperature with fan (appx. 30 min)
3) Fix dry tissue in pre-chilled Methanol (-20^C) for 10 min.
4)  Proceed blocking & staining ........
You should have no problem with degradation if you freeze your tissue as rapidly as possible.
"Try fixing with difference fixative if Methanol doesn't work for your IHC
(ie. pre-chilled; Acetone, Acetone:Methanol, Alcohol, 4% PFA, formalin, & etc).

Offline anwaar

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IF on frozen mouse tissue
« Reply #3 on: May 29, 2005, 10:38:58 PM »
Thanks for useful suggestions. Any tips for reducing background? I am using gamma H2AX antibody to detect foci, indicative of DNA double strand breaks, in the liver and kidney tissue. My secondary antibody is labelled with Alexa 488 (for green) and 594 (for red). I see a lot of background even with DAPI only in the green and red channels. This makes identification of foci difficult in the nucleus. However when I use cultured cells there is no background and foci are clearly visible. Thanks a lot for the help.
Anwaar

Offline ImmunoNYC

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IF on frozen mouse tissue
« Reply #4 on: June 02, 2005, 12:23:57 AM »
Anwaar,

Are your sections which are giving you background fixed in PFA or another aldehyde based fixative? If so this is likely causing your background and you can use glycine, lysine etc to block free aldehydes thereby reducing background.

Otherwise what blocking steps are you doing? Are you blocking before adding primary with serum from the same species as your secondary? Have you titrated your primaries and secondaries out sufficiently? Are you diluting your primary and secondaries into an antibody diluent (usually some variation of your block)?

Offline anwaar

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IF on frozen mouse tissue
« Reply #5 on: June 02, 2005, 03:59:29 PM »
Hi MaximinaNYC,

My sections are NOT fixed with PFA. I fix with in cold acetone or methanol. I do use and glycine to block, however, background that I am seeing is with or without using any antibody i.e. with DAPI alone as well.
Thanks.
Anwaar

Offline ImmunoNYC

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IF on frozen mouse tissue
« Reply #6 on: June 02, 2005, 05:06:22 PM »
Oh sorry, I forgot that you mentioned you do acetone for 30 min. Definitely change the way you are freezing, fixing tissue, this slow freeze and enormously long acetone fix is the likely culprit in all this.

Also, for nuclear antigens on frozens I never use acetone, so you may consider trying a fix in 2% PFA for 10 minutes instead of acetone.

One more Q: how thick are your sections?

Kidneys and liver do have a high autofluorescence inherent to them so you may end up needing to do a more sophisticated block of autofluorescence using sudan black or other methods but i would only do this if you reeeeeeeeally think you have to after optimizing your freezing, fixation protocol. Let me know if you need references.

Offline madeleine huey

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Hi autofluorescence
« Reply #7 on: June 17, 2005, 07:17:14 PM »
Oh, yes.  I understand your pain.  Here's some of my suggestion for changes .  I also use Alexa 488 & 595 as 2nd Ab & couterstain with DAPI;
Dry frozen section @ 50C for 20 min, wash 3x PBS
1) Pre-fix with pre-chilled -20C Acetone:Methanol (50:50) @-20C for 10 min.
"most my ab work with this fixation", fan dry for 1'-5', wash 3x PBS
2) Block with 10% normal goat serum +0.1% Tx100+0.2%NaN3/PBS.  
3) Incubate 1st Ab/2%BSA+0.1%Tx100+0.2%NaN3/PBS
Wash 3x PBST (10'ea)
4) Incubate 2nd Ab/TBST for 2 - 3 hr with (1:800 - 1:1000)
5) Wash 3x PBST (10'ea)
6) After 2nd Ab, quench with 0.3M Glycine/ddH20 for 30' or more "test your tissue's end point".
7) then couterstain with VectaShield+DAPI, & mount with coverslip
*you may ommit NaN3 in ab diluent (I make this buffer for frozen & parraffin interchangeable).
The above protocol work very well for most my mouse tumor and other tissues.  I am trying to block the Kidney & Liver next week.  
Good LucK!

Offline ImmunoNYC

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IF on frozen mouse tissue
« Reply #8 on: June 18, 2005, 12:52:35 AM »
Is your secondary cross adsorbed against mouse? This might explain the autofluorescence. I find Molecular Probes Secondaries are less than optimal for frozen tissues as opposed to cells grown on slides.

Offline madeleine huey

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Block End. Biotin
« Reply #9 on: June 29, 2005, 04:01:12 PM »
My background for my frozen tumors, liver, & kidney frozen tissue.
1)Pre-fix with 4% PFA (5')
2)3x wash
3) 0.3M Glycine (10'-35')
4)3x wash
5) 2 egg white mix in 200 ml ddH20 for 1 hr
6) 3x wash
7) 5% non fat milk/0.5% Tx100/0.05% Tw20/0.2% NaN3 for O/N @ 4C
8) Incubated 1st ab in 1% non fat milk/0.5% Tx100/0.05% Tw20/0.2% NaN3.......( Triton X100 from could be 0.05% - 1%, depending on your target protein) continue your usual protocol.

Block End. Biotin
« Reply #9 on: June 29, 2005, 04:01:12 PM »