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thick slice IHC help!!!

thick slice IHC help!!! - Immunohistochemistry Forum

thick slice IHC help!!! - Immunohistochemistry Forum. Post and discuss questions on immunohistochemistry methods including paraffin, frozen, or free floating tissue sections.


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  #1  
Old 07-23-2010, 05:44 PM
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Exclamation thick slice IHC help!!!



hey,
i'm a summer student and the project I was given was to optimize the immunohistochemistry in my lab. BUT I'M NOT GETTING ANYTHING TO WORK!! I've been told it always worked before....So I was wondering if anyone has any ideas or protocols that work? I'd really appreciate any help I can get!
Here's the protocol I'm using:
our slices are 250-300 um thick.
1) I leave them in formalin for 2 days for the fixative step.
2) Wash slices 3X (15 min each) in PBS-T (PBS with 0.3% tritonX100 to help permeabilize my slices since they're thick)
3) Blocking step for 1/2 hr with 2%normal horse serum in PBS-T
4) Incubate in 1o antibody at 1:1000 in PBS-T + 2%normal horse serum for 72 hrs at 4C with shaking
5) Wash slices 5X in PBS-T
6) Blocking step for 1/2 hr with 2%normal horse serum in PBS-T
7) Incubate at room temp with 2o antibody at 1:500 +2%normal horse serum in PBS-T for 2hrs with shaking and kept in the dark
8 ) Wash 5X (1/2 hr each) in PBS before mounting
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Old 07-23-2010, 06:21 PM
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Default Re: thick slice IHC help!!!

It would help if you gave more details as to how it is not working. Are you getting no staining, too much background, staining in the wrong place, etc? Have you tested your primary/secondary antibodies to make sure they are working? What is the secondary labeled with?

Bryan
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Old 07-27-2010, 04:15 PM
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Default Re: thick slice IHC help!!!

This is my first time doing IHC. I'm a 3rd year undergrad summer student and I've been in my current lab since April.
my primary antibody is from Abcam: Ms mAb to CamKII aplha [6G9] (1.17mg/ml)
ab 2725, lot 864380
recieved April 16, 2010
my secondary antibody is from Invitrogen: Alexa Fluor 488 goat anti mouse IgG (H+L) (2mg/ml)
A11001, lot 745480
The Formalin was ordered last month from Sigma and the PBS is made up fresh every 2 weeks. I make up the PBS-T the day I start a new IHC.
I've been working to optimize the protocol I was given since last month. Since then we have switched from Paraformaldehyde to Formalin as fixative, which seemed to improve the preservation of tissue morphology and actually led to some staining showing up. I've done control slices with secondary only. I also tried a variety of concentrations for primary and different lengths of time in secondary. I found that primary 1:1000 worked well and secondary 1:500 for 2hrs worked better than secondary overnight because otherwise the background staining was too overwhelming. However when I ran the exact same experiment last week NOTHING showed up. Just background and not even the faintest cell staining. I'm confused my this lack in consistency. It just doesn't make any sense to me because it wasn't that the slices had been in fixative too long because the DAPI staining was beautiful as usual on my slices in the recent failed IHC (the DAPI staining is from the Prolong Gold from Invitrogen).
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Old 07-28-2010, 01:34 PM
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Default Re: thick slice IHC help!!!

Oh great, an intermittent failure - the hardest problem to resolve. A few questions, followed by a few ideas:

Q's:
1) What is the tissue source (human, mouse, etc).
2) How is the section prepared (frozen, paraffin, etc)

Ideas:
1) Over fixing can denature antibody-recognized antigens while leaving other stainable features (i.e. DNA) intact. You may want to play with your fixing conditions. Also, have you tried fixing in the cold (4C)?
2) You may want to block using 3% serum in PBS-BSA-NaN3 for 2 hours prior to adding antibody.
3) We permeabilize using 0.05M TBS pH7.5, with 0.01% Tween 20. This may work better than triton, as tween20 isn't as likely to extract proteins.

If you continue having problems, you can get kits which allow you to directly label your primary antibody. This allows you to avoid the secondary, although it makes thing more pricey.

Also, we prefer to buy secondaries from Jackson Immunotech. They have cross-absorbed secondaries, which means that their antibodies are confirmed to bind only to the species in question. This can greatly reduce background. You may want to check out their secondaries.

Bryan
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Old 07-17-2011, 03:09 PM
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Default Re: thick slice IHC help!!!

Hey, take 40 micron sections, while taking sections reduce the speed of the blade movement, embed the brains in the 3% agarose, don't use secondary more than 2 hr(1:500 dil), i should work

Madhu
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